3. Receptors in Airway Disease Beta-adrenoceptors in Lung Inflammation1.2

FRANS P. NIJKAMP and PAUL A. J. HENRICKS

Introduction Airway inflammation is associated with a number of characteristic features like disruption of the epithelial lining and anatomy of the lung, mucus secretion and mucosal edema, increase in inflammatory cells, and nonspecific hyperreactivity of the airways. The latter is the most common characteristic of bronchial asthma. Nonspecific airway hyperreactivity can be defined as an increased reactivity ofthe respiratory airways to physical, chemical, and pharmacological stimuli. Several abnormalities have been postulated to underlie the hyperreactivity, such as beta-adrenoceptor dysfunction, hyperresponsiveness of airway smooth muscle, epithelial damage or dysfunction, and increased reflex constriction of the airways. Resident cells, like epithelial cells, mast cells, and macrophages, may induce inflammation after stimulation and attract other inflammatory cells (neutrophils, eosinophils, monoeytes, lymphocytes) to the lungs, which amplify the inflammatory process. Resident and inflammatory cells may as a result of the inflammation rapidly generate a number of mediators like prostaglandins, leukotrienes, platelet-activating factor (PAF), cytokines, reactive oxygen species, and hydrolytic enzymes. These mediators playa role in the modulation of airway reactivity. There are a number of conflicting reports in the literature about the role of inflammatory cells in the induction of nonallergic airway hyperresponsiveness in relation to autocoid and autonomic receptor dysfunction. The number of inflammatory cells is markedly increased in the respiratory airway of humans dying in status asthmaticus and in mild asthmatics (1). However, hyperresponsiveness persists for several years in asthmatic patients, even in the absence of symptoms, sputum purulence, or other signs of mucosal inflammation (2). The role of inflammatory cells in the induction of airway hyperresponsiveness is difficult to interpret on the basis of contradictory findings in the literature. The autonomic nervous system regulates airway smooth muscle tone and may influence secretion from submucosal glands, transport of fluid across airway epithelium, permeability and blood flow in the bronchial circulation, and release of mediators from inflammatory cells (3). Beta-adrenoceptors are localized on several cell types within lung tissue. It has been possible to demonstrate the coexistence of both beta.- and betas-receptor subtypes in the lung. Beta-edrenoceptors are

SUMMARY Beta-adrenoceptor dysfunction and increase in airway reactivity can be induced by administration of gram-negative bacteria, endotoxin, viruses, and allergens in laboratory animals. However,the deterioration of lung beta-adrenoceptor function is not invariably associated with lung inflammation. Severe asthmatics, but not asthmatics per se, show a diminished beta-adrenoceptor function of airway smooth muscle. These changes are probably a consequence of the active disease state rather than an intrinsic component of asthma. Mediators released from inflammatory cells such as reactive oxygen species and fatty acid metabolites may directly or indirectly induce betaadrenoceptor dysfunction. Beta-adrenoceptor function of leukocytes from asthmatic patients can be decreased as well and it is suggested that Iymphokines like interleukin-2 and interferon-gamma may affect beta-adrenoceptor function. A disturbed beta-adrenoceptor function on inflammatory cells themselves may have consequences for their immune function, mediator release, and effect on surrounding tissues. AM REV RESPIR DIS 1990; 141:5145-$150

physiologically activated by noradrenaline released by sympathetic nerves and beta.-adrenoceptors by the circulating adrenal catecholamine adrenaline. Intact beta-adrenoceptors normally serve to maintain bronchodilatation. Human isolated airway smooth muscle contains a homogeneous population of betasadrenoceptors (4). Besides their presence on airway smooth muscle cells, beta-adrenoceptors are also present on mast cells, epithelial cells, Clara cells, vascular endothelium, vascular smooth muscle, type 2 pneumocytes, submucosal glands, and cholinergic nerves in the airways (5). Stimulation of these receptors leads to inhibition of inflammatory mediator release from mast cells, increased ion and fluid transport by epithelial cells, increased mucociliary clearance, reduced leakiness, smooth muscle relaxation, increased surfactant secretion, increased mucus secretion, and decreased cholinergic transmission. Beta-adrenoceptors have been reported also to be present on inflammatory cells, like lymphocytes, neutrophils, and alveolar macrophages. Stimulation of these receptors leads to decreased immune function and changes in the state of activation of these kind of cells.

Beta-adrenoceptor Function and Asthma In 1968, Szentivanyi (6) postulated that betaadrenoceptor blockade with reduced respiratory beta-adrenoceptor function as a consequence was the fundamental factor in atopic abnormalities in general and bronchial asthma in particular. Since that time the beta-adrenoceptor function in asthmatics has been investigated with a great number of controversial findings (7). A number of investigators have reported evidence for a beta-adrenoceptor deficiency in the lung and other tissues in asthmatic patients. Cerrina and cowork-

ers (8) showed an inverse relationship between in vivo sensitivity to histamine and the in vitro response to isoprenaline, suggesting that betaadrenoceptor function decreases as the level of airway hyperresponsiveness increases. An inverse relationship between beta-adrenoceptor in lymphocytes of asthmatic patients and severity of airway obstruction was also reported (9). Airway smooth muscles removed from severeasthmatics show a decreased betaadrenoceptor function (10). The cellular defect in beta-adrenoceptor function in severe asthmatics patients can also be located somewhere in the subsequent biochemical steps that link the activation of the receptor to final cellular events, i.e.,a defect in the stimulusresponse coupling (11). Chronic administration of beta-adrenoceptor agonists has also been shown to cause reduced beta-adrenoceptor function in the airwaysin man (12). However, beta-adrenoceptor function in asthmatics is relatively resistant to desensitization following exposure to betasagonists (13). Meurs and colleagues (14) showed that beta-adrenoceptor number and function in asthmatic patients are impaired only 24 h after allergen challenge. The allergen challenge induces uncoupling and downregulation of beta-adrenoceptors and nonspecific refractoriness of adenylate cyclase. Lymphocyte membranes from allergic asthmatic patients have a normal beta-adrenoceptor density and a normal adenylate cyclase activity when tested in a stable nonacute phase 1 From the Department of Pharmacology, Faculty of Pharmacy, University of Utrecht, The Netherlands. 2 Correspondence and requests for reprints should be addressed to F. P. Nijkamp, Department of Pharmacology, Catharynesingel 60, 3511 GH Utrecht, The Netherlands.

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of the disease despite the presence of an increased airway reactivity to histamine or acetylcholine (11). Therefore, the changes in beta-adrenoceptor function in asthmatic patients are probably a consequence of an active disease state rather than an intrinsic component of asthma. The hypothesis of Szentivanyi (6) is based on an analogy between the symptoms of patients with bronchial asthma and those symptoms that could be induced by intraperitoneal administration of Bordetella pertussis in experimental animal models. Administration of the bacteria to the animals induces hypersensitivity to bronchospasmic agents (e.g., histamine), an altered immunological reaction to antigens, and a decreased response to betaadrenoceptor agonists. Decreased l3-adrenoceptor numbers and function have also been observed with other gram-negative bacteria (15-17). Downregulation of the beta-adrenoceptor by circulating catecholamines is not involved in this animal model (17, 18). The beta-adrenoceptor dysfunction is accompanied by an increased number of eosinophils in the blood and an increased sensitivity to produce IgE antibodies (19).The common cell wall component of the gram-negative bacteria, endotoxin, is responsible for some of these findings (16). The beta-adrenoceptor dysfunction, however, does not seem to be associated with lung inflammation in this model. Induction of Airway Hyperreactivity and Beta-adrenoceptor Dysfunction There are a number of substances capable of inducing airway hyperreactivity when administered to laboratory animals. Two major groups can be discerned: agents that induce a delayed (> 24 h) increase in airway reactivity and agents that induce a rapid « 24 h) increase in airway reactivity. The rapid increase in airway hyperreactivity by the latter group is accompanied by an inflammatory reaction. This group comprises noxious gases, cigarette smoke, C. a, and inhaled or intravenously administered bacterial endotoxin. The hyperreactivity is thought to result from the inflammatory reaction and to be mainly due to increased reflex bronchoconstriction caused by damage of airway epithelium. No beta-adrenoceptor dysfunction has been reported. The group of substances that induce a delayed (> 24 h) increase in airway reactivity comprises viruses, intraperitoneally injected endotoxin and gram-negative bacteria, parasites, and allergens. All these substances induce or enhance IgE antibody production in addition to an increase in the airway smooth muscle reactivity, suggesting that these two phenomena are related. As far as tested, a beta-adrenoceptor dysfunction has been found. Viral infections have been shown to be of more importance than bacterial infections in the pathogenesis of asthma. Viral respiratory infections can cause airway hyperreactivity in normal and asthmatic subjects (20). Experimental airway infection with parainfluenza type-3 virus induces a hyperreactivity

NIJKAMP AND HENRICKS

of the airways in vivo four days after intranasal inoculation in laboratory animals. Furthermore, it has also been shown that respiratory viral infections diminish leukocyte betaadrenoceptor function in asthmatics (20). The beta-adrenoceptor function on mast cells is also decreased after parainfluenza type-3 viral infection. In sensitized dogs, hyperreactivity following allergen challenge is already present before the inflammatory reaction (21). It has also been shown that an increase in the number of inflammatory cells, accompanied by injury of the guinea pig respiratory airways, caused airway hyporeactivity rather than hyperreactivity and no changes in beta-adrenoceptor function (22). Moreover, hyperreactivity is not invariably seen in lung diseases such as cystic fibrosis, chronic bronchitis, and the adult respiratory distress syndrome, in which the airways are typically infJltrated with inflammatory cells (23). It cannot be excluded that hyperreactivity and beta-adrenoceptor dysfunction can be induced after specific stimulation of inflammatory cells. Inflammatory cells may release mediators like eicosanoids, PAF, hydrolytic enzymes, oxygen radicals, and cytokines, which interact in a complex way to induce changes in receptor function. Beta-adrenoceptors and Immune Cells Inflammatory and immune responses of cells are related to intracellular changes of cyclic AMP levels. In general, increase of cyclic AMP levels induced by beta-adrenoceptor agonists, prostaglandin E 2 , or phosphodiesterase inhibitors diminishes the responsiveness ofimmune cells. Beta-adrenoceptors have been reported to be present on several types of immune cells and many investigators have used human leukocytes isolated from peripheral blood as an in vitro model to study and characterize beta-adrenoceptor function in asthma (19). The density of beta-adrenoceptors in lymphocytes of asthmatic patients not receiving beta-agonist therapy can be substantially decreased (24). One has also to consider that changes in receptor number and function on leukocytes may have consequences for their cellular function itself. As we have pointed out before, the beta-adrenoceptor dysfunction is probably not a fundamental abnormality in asthma but may be acquired. A number of studies have demonstrated a disturbance of cell-mediated immunity in asthma. Lymphocytes are conspicuous among the inflammatory cells infiltrating the bronchi in asthmatic patients. A relative deficiency in the number of T-suppressor cells has been observed in both intrinsic and extrinsic asthma. This indicates that an abnormality in the regulation of T-Iymphocytes and the lymphokines they liberate can be expected. Recently it has been shown that T-helper cells are activated in acute severeasthma (25). Also, an inhibitory influence of beta-adrenoceptor agonists on lymphocyte activation and production of Iymphokines by these cells has been reported (26).

Besides decreased beta-adrenoceptor number and function in the airways of guinea pigs induced by intraperitoneal administration of endotoxin, the number of beta-adrenoceptors on splenic lymphocytes and the beta-adrenoceptor agonist-induced increase in cyclic AMP in these lymphocytes is also diminished in this animal model (27). Surgical removal of the spleen prevents the endotoxin-induced beta-adrenoceptor impairment in the airways (figure 1). AdditionalIy, the T-Iymphocyte selective immunosuppressive drug cyclosporin A prevents the decreased beta-adrenoceptor agonist-stimulated cyclic AMP production in lymphocytes after endotoxin injection (figure 2) (28). These data suggest that T-Iymphocytes ofthe Iymphokines produced by these celIsmay affect the beta-adrenoceptor function. In accordance herewith, it has been reported that immunization of mice with a T-celI-dependent antigen reduced the number of beta-adrenoceptor binding sites on lymphocytes at 1to 5 days postimmunization (29). Several gram-negative bacteria and endotoxin increase the cell number, proliferation, and interleukin-2 production in popliteal lymph nodes of mice and rats (30). Some lymphokines (interleukin-2 and interferon-y) can inhibit agonist-stimulated cyclic AMP production in lymphocytes (31,32). Interestingly, Gulick and coworkers (33) demonstrated that a soluble product of activated lymphocytes

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could inhibit beta-adrenoceptor agonist-stimulated increase in the intracellular concentration of cyclic AMP in other cell types. Interferon production is induced by viruses that cause respiratory tract infections. Influenza infections increase the production of interferon-y and interleukin-Z after allergen stimulation of lymphocytes obtained from asthmatics (34). The effect of interleukin-Z on cyclic AMP production in cells is mimicked by phorbolesters (35). These substances cause translocation of protein kinase C to the plasma membrane and the phosphorylation of various sites in the beta-adrenoceptor-adenylate cyclase system. Protein kinase C activation is involved in numerous cellular activation processes via stimulation of the receptor-coupled phosphatidylinositol turnover. It has been reported that beta-adrenoceptor desensitization is correlated to phosphorylation of the receptor (36). It has to be investigated whether the lymphokines produced during activation of lymphocytes might affect the beta-adrenoceptor function in other tissues as the respiratory airways when these lymphokines are released in the circulation. With regard to this hypothesis, it is relevant to note that the beta-adrenoceptor dysfunction in asthmatic patients is not limited to leukocytes and airways since decreased metabolic, cardiovascular, and eosinopenic responses to beta-adrenoceptor agonists are observed as well. Besides betaadrenoceptor impairment, the function of other receptors coupled to adenylate cyclase, such as the prostaglandin E z and histamine Hz receptors, are also decreased in leukocytes of asthmatic patients (11). It cannot be excluded that these defects may also be medi-

ated by interleukin-Z since this Iymphokine affects the catalytic activity of adenylate cyclase. Neutrophils can be detected in the lung tissues during infections or inflammatory reactions in the lung. The activity of neutrophils is depressed by incubating these cells with beta-adrenoceptor agonists (39,40). Virustreated neutrophils become less responsiveness for isoprenaline (20). Beta-adrenergic blockade augments microvascular injury in an in vivo model of neutrophil-dependent inflammation (41). Because catecholamines can reduce neutrophil-provoked inflammation by inhibiting lysosomal enzyme release and oxygen radical production, an impairment in beta-adrenoceptor function might be translated into increased tissue inflammation and thereby airway hyperreactivity, Beta-adrenoceptors have been reported also to be present on alveolar macrophages of guinea pigs (37) and humans (38). The betaadrenoceptors on alveolar macrophages are not involved in the release of reactive oxygen species of lysosomal enzymes by these cells. More studies are needed to elucidate the function of the beta.-adrenoceptor on alveolar macrophages. Mast cells and their circulating analogues, the basophils, play an important role in the immediate hypersensitivity reaction because the release of mast cell mediators may explain several features of airway hyperreactivity (42). Attempts to determine how mast cell function may be regulated have included the identification of several receptors, among them receptors for betaj-adrenergic agents. These adrenoceptor agonists inhibit the release of, e.g.,histamine by mast cells. However, the latephase response to allergen and the subsequent airway hyperresponsiveness are not prevented by betaj-adrenoceptor agonists (42).

Inflammation and Reactive Oxygen Species Alveolar macrophages are the primary defenders in the lung against inhaled particles and microorganisms. Phagocytic cells like neutrophils and monocytes also accumulate in the lung compartments during bacterial and viral infections in the airways. These cells possess the most potent physiological system to produce reactive oxygen species. The oxygen species are important components of the oxygen-dependent killing of microbes by phagocytes. However, because these products are also released in the extracellular space, tissue injury can take place. The oxygenderived molecules include superoxide anions, hydrogen peroxide, and hydroxyl radicals. These molecules are characterized by an increased reactivity compared with oxygen towards biomolecules like proteins, lipids, and sugars (43). Most receptors including the betaadrenoceptor are part of the membrane and free radicals may damage these membrane constituents. Beta-adrenoceptors contain several disulfide bonds and sulfhydryl groups that may be involved in hormone binding to and acti-

vation ofthe beta-adrenoceptor, Oxygen species are highly reactive with sulfhydryl residues of peptides and proteins. Loesberg and colleagues (44) showed a highly significant positive correlation between the amount of generated superoxide production by alveolar macrophages and the severity of tracheal betaadrenoceptor function deterioration in individual animals (figure 3). It has also been shown that guinea pig alveolar macrophages initiate a specific deterioration of tracheal smooth muscle beta-adrenoceptor function when appropriately stimulated (45). The in vitro system comprises attachment of macrophages to an isolated tracheal spiral from a guinea pig (figure 4). The close proximity of the alveolar macrophages to the tracheal preparation favors a possible influence of short-lived mediators from phagocytic cells on tracheal tissue. By using scavengers and inhibitors of activated oxygen metabolites, it was indirectly shown that the attenuation of 13-adrenoceptor was due to hydroxyl radicals produced by the alveolar macrophages (table I) (45). Moreover, 13-adrenoceptor density is decreased both in vitro and in vivo in the lungs of rats after incubation with oxygen radical-generating systems (46).

Lipid Membrane Properties and Beta-adrenoceptor Function It is well documented that the beta-adrenoceptor-adenylate cyclase complex is extremely sensitive to changes in its lipid environment. This sensitivity to membrane properties is thought to derive from the fact that some or all of the components of hormone-sensitive adenylate cyclase complex undergo some lateral diffusion in the membrane during the process of transmembrane signaling. The

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5148

NIJKAMP AND HENRICKS

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transduction system through which binding of an agonist to the beta-adrenoceptor leads to the cellular response consists of the enzyme adenylate cyclasegenerating cyclicAMP and a regulatory G protein. This multicomponent system consists of at least three distinct inte-

gral proteins distributed asymmetrically in the plasma membrane and is sensitive to changes in membrane fluidity (47). It was demonstrated that dietary linoleic acid modulates guinea pig tracheal betaadrenoceptor responsiveness to the same ex-

TABLE 1 INFLUENCE OF INHIBITORS ON GUINEA PIG ALVEOLAR MACROPHAGE-INDUCED DECREASE OF TRACHEAL RELAXATION TO ISOPRENALINE" Treatment

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" Adapted from Engels and colleagues (45). Tracheal preparations with or without alveolar macrophages were incubated for 1 h with serum obtained from animals treated intraperitoneally with Hsemophilus influenzas 4 days prior to the experiments. Catalase (5,000 U/ml), thiourea (25 mM), and superoxide dismutsse (300 U/ml) were added to the cells 30 min before serum stimulation. The tracheal preparations were precontracted with carbachol before the isoprenaline dose-response curve were made. t p < 0.002 compared to no alveolar macrophages (Student's t test).

tent as in an animal model of atopy (48). Alterations in membrane fluidity might have been the mechanism of action of this betaadrenoceptor attenuation. This hypothesis is supported bydata demonstrating that optimal tracheal beta-adrenoceptor relaxation and highest number of beta-adrenoceptor binding sites in splenic lymphocytes coincide with the highest fluidity of the lymphocyte membranes and vice versa (49). Alterations in membrane fluidity can also be caused by lipid peroxidation-inducing changes in phospholipid bilayer rigidity. In particular, peroxidation of membrane phospholipids, a process that has been associated with a variety of pathological states, like inflammation, results in a loss of beta-adrenoceptor binding sites (46). This process may be initiated through the generation of reactive oxygenspeciesby phagocytic cellsor from free unsaturated fatty acids, which are the result of the catalytic action of phospholipase A z or phospholipase C. Lipid Mediators Alveolar macrophages from asthmatic patients release greater amounts of lipid mediators, such as thromboxanes, prostaglandins, and PAF,than those derived from normal subjects (42). Phospholipase A, is a membranebound enzyme that catalyzes the mobilization of arachidonic acid and PAF from membrane phospholipids. Phospholipase Az appears to play an important role in inflammatory reactions in the airways and beta-adrenoceptor dysfunction (50). During inflammatory and anaphylactic reactions of beta-adrenoceptor stimulation (51,52), the phospholipase A z is strongly activated. After liberation from the membrane phospholipids, arachidonic acid can be converted by specific lipoxygenases into leukotrienes, lipoxines, and several hydroxy acids (HETEs) or by cyclooxygenaseinto prostaglandins and thromboxanes. The initial products generated from arachidonic acid are hydroperoxyeicosatetraenoic acids (HPETEs). 15-HPETE has been shown to reduce beta-adrenoceptor binding sites and function in the lung after in vitro or in vivo administration (53,54). Interestingly,the 15-HETE is the most common derivative of arachidonic acid in the lung (55). During the reduction of the hydroperoxide to the hydroxy form in the arachidonic acid cascade, an oxidant reactant is released that may contribute to the loss of beta-adrenoceptor function and binding sites. The identity of the oxidizingagents is not known; they may be hydroxyl radicals, singlet oxygen, or superoxide anions. The evidence for any biologi.. cal activity of oxygen radicals is mainly based on the use of antioxidants since most of the species are too ephemeral to be measured in in vitro systems (43). The prostaglandins synthesis-inhibitor indomethacin, prevents isoprenaline-induced beta-adrenoceptor desensitization in airways of animals and humans, but does not prevent the decrease in numbers of beta-adreno-

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BETA-ADRENOCEPTORS IN WNG INFLAMMATION

ceptor binding sites (56, 57). In guinea pig trachea, prostaglandin E, reverses the effect of indomethacin in preventing isoprenalineinduced desensitization of the beta-adrenoceptor (58). These results point a possible role of cyclooxygenase products in this type of desensitization. On the other hand, inhibition of the enzyme cyclooxygenase may stimulate the acetylation of free arachidonic acid into the phospholipid pool of the membrane with a change in membrane fluidity as a consequence and subsequent changes in beta-adrenoceptor function. Negative results have been described as well concerning effects of indomethacin on isoprenaline-induced betaadrenoceptor desensitization (59). Anti-inflammatory glucocorticoids, which have been shown to inhibit phospholipase A, activity (51,60), also restoredisease- and betaagonist-induced dysfunction of airway betaadrenoceptors (12, 19). Dexamethasone, an inhibitor of phospholipase A z (60), abolished the detrimental effect of alveolar macrophages on tracheal smooth muscle beta-adrenoceptor function (45). A direct relationship in an animal model of asthma was reported by Taki and associates (50). They showed that the number of beta-adrenoceptors in animals exposed to an aerosol of ovalbumin for 10 successive days was decreased by approximately 40070, while phospholipase activity of lung membranes in the experimental group was elevated by 50%. Also, Barnes and colleagues (61)demonstrated that lung beta-adrenoceptor number after ovalbumin sensitization of guinea pigs was decreased. Phospholipase A,-induced products may induce uncoupling ofthe beta-adrenoceptor and adenylate cyclase by promoting the phosphorylation of the beta-adrenoceptor (57). PAF is a potent biologically active substance that can be synthesized by two distinct synthetic pathways (42). Several characteristic features of asthma can be induced by PAF and it is one of the most potent inducers of airway constriction. It can be produced by a number of inflammatory cells and PAF also activates these kind of cells to release secondary inflammatory mediators, including lipoxygenase and cyclooxygenase products, oxygen radicals, and lysosomal enzymes (42). In vivo, PAF results in the recruitment of various inflammatory cells into tissues following administration (62). PAF is able to elicit a downregulation of beta-adrenoceptors in human lung in vitro (63). Also, a reduced bronchodilator response to isoprenaline is measured in vivo in guinea pigs made hyperresponsive by treatment with PAF (64).However, no changes in beta-adrenoceptor numbers or binding affinity is observed in lungs removed from PAF-treated guinea pigs in comparison with control animals (64). PAF reduces the increase in intracellular cyclic AMP concentrations induced by prostaglandins E 2 and salbutamol in guinea pig alveolar macrophages (65). It is not known whether indirect effects of PAF, like stimulation of oxygen radicals by phagocytic cells, contribute to beta-adrenoceptor desensitization.

Conclusions

Airway inflammation is associated with characteristic features like disruption of the epitheliallining and anatomy of the lung, mucus secretion, mucosal edema, and increased numbers of inflammatory cells. The number of inflammatory cells in the lungs of severe and mild asthmatics is markedly increased. Severeasthmatics, but not asthmatics per se, show a diminished beta-adrenoceptor function of airway smooth muscle. These changes are probably a consequence of the active disease state rather than an intrinsic component of asthma. Beta-adrenoceptor dysfunction and increase in airway reactivity can be induced by administration of gram-negative bacteria, endotoxin, viruses, and allergens in laboratory animals. However, the deterioration of lung beta-adrenoceptor function is not invariably associated with lung inflammation. Beta-adrenoceptor function of leukocytes from asthmatic patients can be decreased as well. It is suggested that T-Iymphocytes or Iymphokines may affect beta-adrenoceptor function. Interleukin-2 and interferon-y can inhibit agonist-stimulated cyclic AMP production in lymphocytes. A disturbed beta-adrenoceptor function on leukocytes may have consequences for their immune function as well. Oxygen-derived products from phagocytic cells may influence beta-adrenoceptors by a direct action on the disulfide bonds and sulfhydrylgroups of these receptorsor by lipid peroxidation of the phospholipid membrane in which the beta-adrenoceptors are present. Reactiveoxygen speciesfrom free polyunsaturated fatty acids may also initiate this process. It is well documented that the beta-adrenoceptor adenylate cyclase complex is sensitive to changes in its lipid environment, i.e., to changes in membrane fluidity. Membrane fluidity can be altered by lipid peroxidation or changes in the diet. References I. Dunnill MS. The pathology of asthma with special references to the changes in the bronchial mucosa. J Clin Pathol 1960; 13:27-33. 2. Townley RG, Ryo UY, Kolotkin BM, Kay B. Bronchial sensitivity to methacholine in current and former asthmatic and rhinitic patients and control subjects. J Allergy Clin Immunol1975; 56:429-42. 3. Nadel JA, Barnes PJ. Autonomic regulation of the airways. Ann Rev Med 1984; 35:451-67. 4. Zaagsma J, Van der Heijden PJCM, Van der Schaar MWG, Bank CMC. Comparison of functional Bsadrenoceptor heterogeneity in central and peripheral airway smooth muscle of guinea pig in man. J Recept Res 1983; 3:89-106. 5. Barnes PJ. Beta-adrenoceptors in lung tissue. In: Morley J, ed. Perspectives in asthma. 2. Betaadrenoceptors in asthma. London: Academic Press, 1984; 67-95. 6. Szentivanyi A. The beta-adrenergic theory of the atopic abnormality in bronchial asthma. J Allergy 1968; 42:203-32. 7. Barnes PJ. Neural control of human airways in health and disease. Am RevRespir Dis 1986;134: 1289-314. 8. Cerrina J, Le Roy Ladurie M, Labat C, Raffestin B, Bayol A, Brink C. Comparison of human bronchial muscle responses to histamine in vivo with

histamine and isoproterenol agonists in vitro. Am Rev Respir Dis 1986; 134:57-61. 9. Brooks SM, McGowan K, BernsteinIL, Altenau P, Peagler J. Relationship between numbers of betaadrenergic receptors in lymphocytes and disease severity in asthma. J Allergy Clin Immunol 1979; 63:401-6. 10. Goldie RG, Spina D, Henry PJ, Lulich KM, Paterson JW.ln vitro responsivenessof human asthmatic bronchus to carbachol, histamine, beta-receptor agonists, and theophylline. Br J Clin Pharmacol 1986; 22:669-76. II. Meurs H, Kauffman HF, Koeter GH, Timmermans A, De Vries K. Regulation of the beta-receptor-adenylate cyclasesystem in lymphocytesof allergic patients with asthma: possible role for protein kinase C in allergen-induced nonspecific refractoriness of adenylate cyclase. J Allergy Clin Immunol 1987; 80:326-39. 12. Holgate ST, Baldwin CJ, Tattersfield AE. I3-Adrenergic agonist resistance in normal human airways. Lancet 1977; 11:375-7. 13. Harvey JE, Tattersfield AE. Airway response to salbutamol: effect of regular salbutamol inhalations in normal, atopic and asthmatic subjects. Thorax 1982; 37:280-7. 14. Meurs H, Keeter GH, De Vries K, Kauffman HE The beta-adrenergic system and allergic bronchial asthma: changes in lymphocyte beta-adrenergic receptor number and adenylate cyclase activity after an allergen-induced asthmatic attack. J Allergy Clin Immunol 1982; 70:272-80. 15. Schreurs AJM, Nijkamp FP. Haemophi/us influenzae induced loss of lung l3-adrenoceptor binding sites and modulation by changes in peripheral catecholaminergic input. Eur J Pharmacol 1982; 77:95-102. 16. Schreurs AJM, Verhoef J, Nijkamp FP. Bacterial cell wall components decrease the number of guinea-pig lung l3-adrenoceptors. Eur J Pharmacol 1983; 127-32. 17. Engels F, Folkerts G, Van Heuven-Nolsen D, Nijkamp FP. Haemophilus injluenzae-induced decreasesin lung l3-adrenoceptorfunction and number coincide with decreases in spleen noradrenaline. Naunyn Schmiedebergs Arch Pharmacol1987; 336:274-9. 18. Schreurs AJM, Versteeg DHG, Nijkamp FP. Involvementof catecholamines in Haemophilus influenzae induced decrease of l3-adrenoceptor function. Naunyn SchmiedebergsArch Pharmacol1982; 320:235-9. 19. Nijkamp FP. Hyperreactivity, inflammation and the l3-adrenoceptor In: Bonta IL, Bray MA, Parnham MJ, eds. Handbook of inflammation. Vol 5. The pharmacology of inflammation. Amsterdam: Elsevier, 1985; 335-54. 20. Busse WW. Infections. In: Barnes PJ, Rodger IW, Thomson NC, eds. Asthma: basic mechanisms and clinical management. London: Academic Press Ltd, 1988; 483-502. 21. O'Byrne PM, Dolovich J, Hargreave FE. Late asthmatic responses. Am Rev Respir Dis 1987;136: 740-51. 22. Folkerts G, Henricks PAJ, Siootweg PJ, Nijkamp FP. Endotoxin-induced inflammation and injury of the guinea pig respiratory airways cause bronchial hyporeactivity, Am RevRespir Dis 1988; 137:1441-8. 23. BousheyHA, Holtzman MJ. Experimental airway inflammation and hyperreactivity. Searching for cell and mediators. Am Rev Respir Dis 1985; 131:312-3. 24. Sano Y, Watt G, Townley RG. Decreased mononuclear cell beta-adrenergic receptors in bronchial asthma: parallel studies of lymphocyte and granulocytedesensitization. J AJlergy Clin Immunol 1983; 72:495-503.

5150 25. Corrigan CJ, Hartnell A, Kay AB. T lymphocyte activation in acute severe asthma. Lancet 1988; 1:1129-32. 26. Paegelow I, Werner H. Influence of adrenergic agonists and antagonists on lymphokine secretion in vitro. Int J Immunopharmacol 1987; 9:761-8. 27. Van Oosterhout AJM, Folkerts G, Ten Have GAM, Nijkamp FP. Involvement of the spleen in the endotoxin-induced deterioration of the respiratory airway and lymphocyte ~-adrenergic systems of the guinea pig. Eur J Pharmacol1988; 147:421-9. 28. Van Oosterhout AJM Woutersen-van Nijnanten FMA, Nijkamp FP. Cyclosporin-A prevents endotoxin-induced l3-adrenoceptor impairment in lymphocytes. Eur J Pharrnacol 1988; 149:191-2. 29. Fuchs BA, Albright JW, Albright JF. ~-Adren­ ergic receptors on murine lymphocytes: density varies with cell maturity and lymphocyte subtype and is decreased after antigen administration. Cell Immunol 1988; 114:231-45. 30. Gearing AJH, Bird C, Wadha M, Redhead K. The primary and secondary cellular immune responses to whole cell Bordetella pertussis vaccine and its components. Clin Exp Immunol 1987; 68:275-81. 31. Beckner SK, Farrar WL. Inhibition of adenylate cyclase by IL 2 in human T lymphocytes is mediated by protein kinase C. Biochem Biophys Res Commun 1987; 145:176-82. 32. Davis VL, Earp HS, Stempel DA. Interferon inhibits agonist-induced cyclic AMP accumulation in human lymphocytes. Am Rev Respir Dis 1984; 130:167-70. 33. Gulick T, Chung MK, Pieper SJ, Schreiner GF, Lange LG. Immune eytokine inhibition of betaadrenergic agonist stimulated cyclic AMP generation in cardiac myocytes. Biochem Biophys Res Commun 1988; 150:1-9. 34. Lin C-Y, Kuo Y-C, Liu W-T, Lin c-c. Irnmunomodu1ation of influenza virus infection in the precipitating asthma attack. Chest 1988; 93:1234-8. 35. Yamashita A, Kurokawa T, Une Y, Ishibashi S. Phorbol ester regulates stimulatory and inhibitory pathways of the hormone-sensitive adenylate cyclase system in rat reticulocytes. Eur J Pharmacol 1988; 151:167-75. 36. Sibley DR, Strasser RH, Benovic JL, Daniel K, Lefkowitz RJ. Phosphorylation/dephosphorylation of the ~-adrenergicreceptor regulates its functional coupling to adenylate cyclase and subcellular distribution. Proc Nat! Acad Sci USA 1986; 83: 9408-12. 37. Henricks PAJ, Van Esch B, Van Oosterhout AJM, Nijkamp FP. Specific and non-specific effects of ~-adrenoceptoragonists on guinea pig alveolar macrophage function. Eur J Pharmacol 1988; 152:321-30. 38. Beusenberg FD, Adolfs MJP, Van Schaik-van Groningen JME, Hoogsteden HC, Bonta IL. Regu-

NIJKAMP AND HENRICKS

lation of cyclic AMP levels in alveolar macrophages of guinea pigs and man by prostanoids and l3-adrenergic agents. Agents Actions 1989; 26:105-7. 39. Mack JA, Nielson CP, Stevens DL, Vestal RE. J)Adrenoceptor-mediated modulation of calcium ionophore activated polymorphonuclear Ieucocytes, Br J Pharmacol 1986; 88:417-23. 40. Tecoma ES, Motulsky HJ, Traynor AE, Omann GM, Muller H, Sklar LA. Transient catecholamine modulation of neutrophil activation: kinetic and intracellular aspects of isoproterenol action. J Leukocyte Bioi 1986; 40:629-44. 41. Weisdorf OJ, Jacob HS.I3-Adrenergic blockade: augmentation of neutrophil-mediated inflammation. J Lab Clin Med 1987; 109:120-6. 42. Barnes PJ, Chung KF, Page CPo Inflammatory mediators and asthma. Pharmacol Rev 1988; 40:49-84. 43. Nijkamp FP, Henricks PAJ. Free radicals in pulmonary disease. In: Barnes PJ, Rodger IW, Thomson NC, eds. Asthma: basic mechanisms and clinical management. London: Academic Press Ltd, 1988; 315-23. 44. Loesberg C, Henricks PAJ, Nijkamp FP. Inverse relationship between superoxide anion production of guinea pig alveolar macrophages and tracheal Bsadrenergic receptor function; influence of dietary polyunsaturated fatty acids. Int J Immunopharmacol 1989; 11:165-71. 45. Engels F, Oosting RS, Nijkamp FP. Dual effects of Haemophilus influenzae on guinea pig tracheal beta-adrenergic receptor function: involvement of oxygen-centered radicals from pulmonary macrophages. J Pharmacol Exp Therap 1987; 241: 994-9. 46. Kramer K, Rademaker B, Rozendal WHM, Timmerman H, Bast A. Influence of lipid peroxidation on l3-adrenoceptors. FEBS Lett 1986; 198: 80-4. 47. Levitzki A. I3-Adrenergic receptors and their mode of coupling to adenylate cyclase. Phys Rev 1986; 66:819-54. 48. l..oesberg C, Folkerts G, Nijkamp FP. Effects of dietary linoleic acid on beta-adrenergic responsiveness of the guinea pig respiratory system. Prostagl Leuk Ess Fatty Acids 1988; 34:127-34. 49. Loesberg C, Van der Stelt M, Hooyrnan GJ, Hensen EJ, Nijkamp FP.B-Adrenergic binding sites and membrane fluidity of guinea pig lymphocytes. Agents Actions 1989; 26:55-6. 50. Taki F, Takagi K, Satake T, Sugiyama S, Ozawa T. The role of phospholipase in reduced beta-adrenergic responsiveness in experimental asthma. Am Rev Respir Dis 1986; 133:362-6. 51. Blackwell GJ, Flower RJ, Nijkamp FP, Vane JR. Phospholipase A, activity of guinea pig isolated perfused lungs: stimulation and inhibition by anti-inflammatory steroids. Br J Pharmacol1978; 62:79-89. 52. Suzuki K, Sugiyama S, Takagi K, Satake T, Ozawa T. The role of phospholipase in ~-agonist-

induced down regulation in guinea pig lungs. Biochem Med Metabol Bioi 1987; 37:157-66. 53. Folkerts G, Nijkamp FP, Van Oosterhout AJM. Induction in guinea-pigs of airway hyperreactivity and decreased lung l3-adrenoceptor number by 15-hydroperoxy-arachidonic acid. Br J Pharmacol 1983; 80:597-9. 54. Nijkamp FP, Van Oosterhout AJM. Influence of 15-hydroperoxy-arachidonic acid on lung betaadrenoceptor function and airway reactivity. Agents Actions 1984; 15:85-6. 55. Hamberg M, Hedqvist P, Radegran K. Identification of 15-hydroxy-5,8,l1,13-eicosatetraenoic acid (l5-HETE) as the major metabolite of arachidonic acid in human lung. Acta Physiol Scand 1980; 110:219-21. 56. Omini C, Abbracchio MP, Coen E, Daffonchio L, Fano M, Cattabeni F. Involvement of arachidonic acid metabolites in l3-adrenoceptor desensitization: functional and biochemical studies. Eur J Pharmacol 1985; 106:601-6. 57. Abbacchio MP, Daffonchio L, Omini C. Arachidonic acid metabolites and lung l3-adrenoceptor desensitization. Pharmacol Res Commun 1986; 18:93-110. 58. Berti F, Daffonchio L, Folco GC, Omini C, Vigano T. Desensitization of B-adrenoceptors in guinea-pig trachea. A prostaglandin mediated phenomenon. J Auton Pharmacol 1982; 2:247-53. 59. Fernandes LB, Knight DA, Rigby PJ, Spina D. Paterson JW. Goldie RG. I3-Adrenoceptor desensitization in guinea-pig isolated trachea. Eur J Pharmacal 1988; 157:135-45. 60. Nijkamp FP, Flower RJ, Moncada S, Vane JR. Partial purification of rabbit aorta contracting substance-releasing factor and inhibition of its activity by anti-inflammatory steroids. Nature 1976; 263:479-82. 61. Barnes PJ, Dollery CT, MacDermott J. Increased pulmonary a-adrenergic and reduced ~-adrenergic receptors in experimental asthma. Nature 1980; 285:569-71. 62. Archer CB, Page CP, Morley J, MacDonald DM. Accumulation of inflammatory cells in response to intracutaneous platelet-activating factor (Paf-acether) in man. Br J Dermatol 1985; 112: 285-90. 63. Agrawal DK, Townley RG. Effect of platelet activating factor on beta-adrenoceptors in human lung. Biochem Biophys Res Commun 1987; 143:1-6. 64. Barnes PJ, Grandordy BM, Page CP, Rhoden KJ, Robertson ON. The effect of platelet activating factor on pulmonary l3-adrenoceptors. Br J Pharmacol 1987; 90:709-15. 65. Bachelet M, Adolfs MJP, Masliah J, Bereziat G, Vargaftig BB, Bonta IL. Interaction between PAF-acether and drugs that stimulate cyclic AMP in guinea-pig alveolar macrophages. Eur J Pharmacol 1988; 149:73-8.