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The roles of protein-tyrosine kinases in neutrophil and airway smooth muscle cell activation Dryden, Peter John 1992

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THE ROLES OF PROTEIN-TYROSINE KINASES IN NEUTROPHIL ANDAIRWAY SMOOTH MUSCLE CELL ACTIVATION: STUDIES USINGPROTEIN-TYROSINE KINASE INHIBITORSbyPETER JOHN DRYDENB.Sc., University of Victoria, 1990A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Experimental Medicine)We accept this thesis as conformingto the required standardTHE UNIVERISITY OF BRITISH COLUMBIAAugust 1992© Peter John Dryden, 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly  purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of 	MedicineThe University of British ColumbiaVancouver, CanadaDate 	 September 11, 1992DE-6 (2/88)Abstract THE ROLES OF PROTEIN-TYROSINE KINASES IN NEUTROPHIL ANDAIRWAY SMOOTH MUSCLE CELL ACTIVATION: STUDIES USINGPROTEIN-TYROSINE KINASE INHIBITORSBy Peter John DrydenAsthma is an inflammatory lung disease involving reversible airwayobstruction. The characteristic features of asthma include eosinophilia, nonspecificbronchial hyperresponsiveness to inhaled spasmogens, airway epithelial damage,mucosal edema, and increased mucosal gland secretion. Two cellular componentsinvolved in this complex respiratory disease are neutrophils which are the majorcontributer to pulmonary inflammation and airway smooth muscle cells that are theultimate responding cells to the released inflammatory mediators. In this study, themechanisms of cellular activation of these two cell types are examined.Cellular stimulation involves receptor-induced activation of intracellularsignalling pathways that are potential sites of intervention and targets for future drugdesign. Protein-tyrosine kinase activity has been demonstrated in neutrophils,although the role of these enzymes in neutrophil activation is not clearly defined.The effect of protein-tyrosine kinase inhibitors were used to determine the site ofaction of this class of compounds that are capable of inhibiting neutrophil activation.The rise in intracellular calcium is an important link between receptoractivation and cellular stimulation in neutrophils. Changes in Fura-2 fluorescence hasbeen used to determine the efficacy of several protein-tyrosine kinase inhibitors todetermine the most potent inhibitor of neutrophil activation. a-Cyano-3,4-dihydroxythiocinnamamide (DHC) was found to be the most active inhibitor and wasselected to be used for further investigations. Other assays used to assess the activityof this drug as an inhibitor of neutrophil activation include measurement ofi isuperoxide production and elastase release. Both these agonist-stimulatedpathophysiological activities were diminished by DHC, suggesting that protein-tyrosine kinase inhibitors have pharmacologically beneficial effects in the control ofneutrophil activation and possibly inflammatory reactions.Protein-tyrosine kinase activation in neutrophils was detected byimmunoblotting using an antiphosphotyrosine specific antibody and decreases inimmunoreactivity were observed in tyrphostin-treated cells. DHC was found to haveno effect on binding affinity or receptor number although decreasedpolyphosphatidylinositol hydrolysis and protein kinase C activation suggesting thatthe site of action was preceding PLC activation.Airway smooth muscle contraction leads to decreased airflow due to airwaynarrowing. A variety of external factors control signal transduction pathways leadingto smooth muscle contraction. Guinea pig airway smooth muscle contraction inresponse to contractile agonists was diminished by a protein-tyrosine kinase inhibitor,suggesting that this class of enzyme has a role in smooth muscle contraction. Onantiphosphotyrosine immunoblots, tyrosine phosphorylated substrates were identifiedin cultured canine airway smooth muscle cells. Also, phosphorylation of a copolymercontaining poly(Glu, Tyr) 4:1 by smooth muscle extracts implies that these cellscontain protein-tyrosine kinase activity that is dependent on magnesium andmanganese. Using anion exchange chromatography, a major peak of protein-tyrosinekinase activity as well as lower activity peaks were obtained from both cytosolic andNP-40 solubilized particulate smooth muscle extracts. Gel filtration chromatographyresolved an -60 kDa cytosolic peak of protein-tyrosine kinase activity and a highmolecular weight (-400-500 kDa) peak was fractionated from NP-40 solubilizedparticulate extracts. Therefore, detection of protein-tyrosine kinase activity inneutrophils and smooth muscle cells provides a potential site to inhibit the activationof these cells and control asthmatic symptoms.iiiTABLE OF CONTENTSPageTitle page 	  iAbstract 	  iiTable of Contents 	 ivList of Tables	viiList of Figures 	 viiiList of Abbreviations 	 xAcknowledgements	  xiCHAPTER 1. INTRODUCTION1.1. WHAT IS ASTHMA? 	   11.2. CELLULAR COMPONENTS IN ASTHMA	 21.2.1. Inflammatory cells: Mast cells, eosinophils, macrophages, platelets,lymphocytes, neutrophils 	 21.2.2. Effector cells: Epithelial cells and smooth muscle cells 	  81.3 CHEMICAL MEDIATORS IN ASTHMA 	  101.4 ACTIVATION AND SIGNAL TRANSDUCTION IN NEUTROPHILS 	  .131.5 PROTEIN-TYROSINE KINASE ACTIVITY IN NEUTROPHILS 	 201.6 PROTEIN KINASES IN SMOOTH MUSCLE CELLS 	 221.7 PROTEIN-TYROSINE KINASE INHIBITORS	241.8 OBJECTIVES 	 27CHAPTER 2. MATERIALS AND METHODS2.1 MATERIALS 	  302.2 PURIFICATION OF NEUTROPHILS AND LYMPHOCYTES 	 31ivv2.3 MEASUREMENT OF NEUTROPHIL ACTIVATION 	 322.3.1. Immunoblotting with antiphosphotyrosine antibodies 	  322.3.2. Platelet-activating factor radioligand binding 	  332.3.3. Polyphosphatidylinositide hydrolysis 	 332.3.4. Calcium mobilization 	  342.3.5. Protein kinase C assay 	 342.3.6. Superoxide production 	 352.3.7. Elastase release 	 362.4. ANALYSIS OF PROTEIN-TYROSINE KINASE ACTIVITY IN AIRWAYSMOOTH MUSCLE CELLS 372.4.1. Canine sensitization and guinea pig tracheal contraction assay . . . . 372.4.2. Canine tracheal smooth muscle cell culture 	  372.4.3. Preparation of smooth muscle cell extracts 	 382.4.4. Protein-tyrosine kinase assay 	 382.4.5. Chromatography of smooth muscle cell extracts 	  392.5. STATISTICAL ANALYSIS 	 40CHAPTER 3. RESULTS3.1. EFFECT OF TYRPHOSTINS ON NEUTROPHIL ACTIVATION 	 413.1.1. Inhibition of human neutrophil protein-tyrosine phosphorylation . . 413.1.2. Effect of DHC on ligand-receptor interactions 	 443.1.3. Effect of DHC on polyphosphatidylinositide hydrolysis 	 473.1.4. Effect of DHC on ligand-induced increases in cytosolic free Ca2+concentration 	 493.1.5. Inhibition of protein kinase C activation 	 513.1.6. Superoxide release 	 553.1.7. Elastase release 	 57vi3.1.8. Kinetics of inhibition of neutrophil activation by DHC 	  .603.2. IDEN INCATION OF PROTEIN-TYROSINE KINASE ACTIVITY INAIRWAY SMOOTH MUSCLE CELLS	633.2.1. Inhibition of airway smooth muscle contraction by tyrphostins . . . 	 633.2.2. Detection of tyrosine phosphoproteins by immunoblotting 	  653.2.3. Phosphorylation of poly(Glu, Tyr) 4:1 by smooth muscle extracts .. 673.3. PARTIAL PURIFICATION AND ANALYSIS OF PROTEIN-TYROSINEKINASE ACTIVITY FROM AIRWAY SMOOTH MUSCLE CELLS . . .683.3.1. Effect of detergent on extraction of protein-tyrosine kinase activity . 683.3.2. Effect of divalent cations on protein-tyrosine kinase activity 	 693.3.3. Thermal stability of protein-tyrosine kinases 	 713.3.4. Anion exchange chromatography of cultured airway smooth muscle cellextracts 	  723.3.5. Gel filtration chromatography of cultured airway smooth muscle cellextracts 	  72CHAPTER 4. DISCUSSION4.1. INHIBITION OF NEUTROPHIL RESPONSES BY TYRPHOSTINS . . .754.2. IDENTIFICATION OF PROTEIN-TYROSINE KINASE ACTIVITY INAIRWAY SMOOTH MUSCLE CELLS 	 804.3. RELATIONSHIP BETWEEN NEUTROPHIL ACTIVATION ANDAIRWAY SMOOTH MUSCLE CONTRACTION 	 83CHAPTER 5. CONCLUSION 	 85BIBLIOGRAPHY 	 87LIST OF TABLESTable 1. Effect of DHC on PAF binding affinity and receptor number in humanneutrophils 	  46Table 2. Kinetic values for inhibition of superoxide production by DHC 	 62vi iviiiLIST OF FIGURESFigure 1. Model of signal transduction leading to neutrophil activation	  17Figure 2. Structures of protein-tyrosine kinase inhibitors	26Figure 3. Possible mechanism of airway smooth muscle cell hyperresponsiveness 28Figure 4. Inhibition of tyrosine phosphorylation in neutrophils stimulated with PAF,LTB4 , and FMLP by DHC 	 43Figure 5. Lack of effect of DHC on PAF binding to human neutrophils	 45Figure 6. Effect of DHC on agonist-induced polyphosphatidylinositide hydrolysis 48Figure 7. Effect of DHC on intracellular calcium release	 50Figure 8. Effect of DHC on PAF-induced activation of cytosolic PKC activity inhuman neutrophils	 53Figure 9. Effect of DHC on PAF-induced activation of particulate PKC activity inhuman neutrophils	 54Figure 10. Effect of DHC on stimulation of superoxide generation from humanneutrophils	  56Figure 11. Inhibition of elastase release from human neutrophils by DHC 	 58Figure 12. Dose-response curve of inhibition of FMLP-stimulated elastase release byDHC 	 59Figure 13. Kinetic analysis of inhibition of FMLP-induced superoxide formation byDHC 	 61Figure 14. Inhibition of guinea pig airway smooth muscle contraction in response tohistamine, LTD4, and carbachol	64Figure 15. Protein-tyrosine phosphorylation of smooth muscle cell proteins detectedby immunoblotting with an antiphosphotyrosine antibody	 66Figure 16. Time course of protein-tyrosine kinase activity in smooth muscle cellextracts 	  67Figure 17. Extraction of particulate protein-tyrosine kinase with detergents	 68Figure 18. Effect of divalent cation concentration on phosphorylation of poly(glu,Tyr)4:1 in cytosolic and NP-40 solubilized particulate extracts 	  70Figure 19. Stability of protein-tyrosine kinase activity after thermal treatment ofcytosolic and particulate extracts prior to protein-tyrosine kinase assay . . .71Figure 20. MonoQ fast protein liquid chromatography of protein-tyrosine kinaseactivity extracted from cultured airway smooth muscle cells 	 73Figure 21. Superose 6 fast protein liquid chromatography of protein-tyrosine kinaseactivity extracted from cultured airway smooth muscle cells 	 74ixLIST OF ABBREVIATIONSBAL 	  bronchoalveolar lavageBmax 	 maximum number of receptors per cellBSA 	 bovine serum albuminDAG 	  diacylglycerolDIPC 	  4-hydroxy-3,5-diisopropylcinnamamideDMSO 	  dimethyl sulphoxideDFP 	 diisopropylfluorophosphateDHC 	  a-cyano-3,4-dihydroxythiocinnamamideDMEM 	 Dulbecco's Modified Eagle MediumEGF 	 epidermal growth factor1-EV 1 	  forced expiratory volume in 1 secondFMLP	formlymethionylleucylphenyalanineFPLC 	 fast protein liquid chromatographyGM-CSF 	 granulocyte-macrophage colony stimulating factorG-protein 	 guanine nucleotide binding proteinHBSS	Hank's balanced salt solutionHETE 	 hydroxyeicosatetraenoic acid[Pi 	 inositol monophosphateIP2 	 inositol bisphosphate1P3 	 inositol trisphosphateLT 	 leukotrieneMLCK 	  myosin light chain kinaseNP-40	 Nonidet P-4002 	  superoxide anionPAF 	 platelet-activating factorPDGF 	 platelet-derived growth factorPG 	 prostaglandinPIP2 	 phosphatidlyinositol bisphosphatePKA 	 CAMP-dependent protein kinasePKC 	 protein kinase CPMSF 	 phenylmethylsulfonyl fluoridePS 	 phosphatidylserineTBS 	 tris-buffered salineTX 	 thromboxaneACKNOWLEDGEMENTSFirst and foremost, I would like to thank Dr. Hassan Salari for supervisionthroughout my research leading to this degree. I am also grateful to the fundingagencies that made my research possible including the Heart and Stroke Foundationfor B.C. and Yukon and the National Centres of Excellence in Respiratory Health.Also, personal support from the Heart and Stroke Foundation for B.C. and Yukonwas gratefully received.I will take this opportunity to also acknowledge generous gifts of 4-hydroxy-3,5-diisopropylcinnamamide and a-cyano-3,4-dihydroxythiocinnamamide from Dr.A.T. Hudson (Wellcome, U.K.) and cleaned canine trachealis from Dr. N.L. Stephens(Manitoba). Valuable technical assistance was received from Sandra L. Howard,particularily in regards to canine smooth muscle cell culture maintenance and organbath studies. Elastase expertise was obtained from Dr. Felix Ofulue (VGH). Dr.Vincent Duronio (Biomedical Research Centre, UBC) provided helpful discussionsand criticisms during my research.I would like to dedicate this thesis to my parents, Marilyn and Rod Dryden.xiCHAPTER 1INTRODUCTION1.1. WHAT IS ASTHMA?Asthma is a chronic respiratory disease that is characterized by both reversibleairway narrowing and airway hyperresponsiveness (American Thoracic Society,1962). The greater degree of bronchial reactivity and the wide variations inresistance to airflow in intrapulmonary airways are important factors that havefundamental roles in the pathophysiology of asthma. Although a definition of asthmahas not been clearly established, in a clinical setting asthma is identified primarilyfrom patient history. As a diagnostic test, a histamine challenge method along withclinical data suggest a provocative concentration causing a 20 % fall in forcedexpiratory volume (I-EV 1) to 8 mg/ml or less is characteristic of asthma (1). A morepractical definition is based on the presence of specific symptoms, such as attacks ofwheezing and dyspnea (shortness of breath). The risk factors for asthma includeallergy, respiratory infection, heredity, exposure to industrial/occupational orenvironmental irritants, chemical ingestion, exercise, and cigarette smoking (2).These factors cause airway obstruction in asthmatics that results from muscle spasm,mucosal edema, musosal inflammation, and mucous secretion. Although asthma wasinitially characterized as an allergic disease, the fact that many patients have noevidence of allergy suggests that multiple mechanisms are involved in the asthmaticresponse.Asthma is the most common chronic childhood disease and a commoncomplaint of adults. The severity of this disease ranges from a minor nuisance to achronic struggle. The onset of asthma can occur at any age, but peak incidence isbefore the age of five years. Although a precise definition of asthma has not been1established, in the U.K., Canada, the United States, Australia, and New Zealandbetween 10 to 20 % of children ages 7 to 10 years had significant recurrentrespiratory symptoms implicating asthma (3). Also, there was an increase in hospitalpediatric admissions for asthma between 1975 and 1985, despite an increase in theuse of anti-asthmatic medications (3). In adults, few studies have investigated theprevalence and characteristics of asthma due to the difficulty in obtaining a randomsample and due to diagnostic confusion because of other respiratory diseases. Adultasthma is affected most significantly by environmental exposure and occupationalexposure to sensitizing agents that induce airway hyperresponsiveness. Asthma israrely fatal because identifiable risk factors have been characterized to manage severeasthma.1.2. CELLULAR COMPONENTS IN ASTHMA.1.2.1. Inflammatory cellsMultiple cell types are involved in the inflammatory response in asthmaticairways involving both the airway epithelium and the unique target cells such ascough receptors, smooth muscle, and glands. The suggestion that inflammation isinvolved in the pathogenesis of asthma was first proposed because of pathologicalfindings in patients that had died from status asthmaticus. These patients had airwayplugging with an inflammatory exudate and an intense mucosal as well assubmucosal inflammatory infiltrate consisting of neutrophils, eosinophils,lymphocytes, and mononuclear phagocytes (4). These cell types have also beenobserved in increased numbers in the sputum (5) and bronchoalveolar lavage (BAL)fluid (6) of patients with asthma. Clinical data also suggests that a relationship existsbetween the development of inflammation and airway hyperresponsiveness. Forexample, viral respiratory tract infection (7), pollutant exposure (8), and occupational2exposure to agents such as toluene diisocyanate (9) have been shown to causeinflammatory cell infiltration into the airway.1.2.1.1. Mast cells.Mast cells in the lung are activated by many agents. IgE-mediated mast cellactivation during antigen exposure is the best understood mechanism that stimulatesthe release of mast cell derived mediators. Neuropeptides, platelet products, andseveral cytokines are also capable of stimulating mast cells (10). Several mediatorsare released from activated mast cells. Both preformed, rapidly released mediators aswell as newly synthesized mediators are released by mast cells that are found liningthe bronchi, bronchioles and in the interalveolar septa. Mast cell activation byantigen stimulates the initial airway response characterized by smooth musclecontraction, increased vascular permeability, hypotension, and increased mucoussecretion. Numerous mediators released by mast cells contribute to the pathogenesisof asthma.In vitro experiments have revealed that many agents are capable ofstimulating mast cells via IgE-dependent mechanisms (11). Mast cell degranulationcauses the release of chemotactic factors for neutrophils and eosinophils, as well asthe release of mediators that induce smooth muscle contraction and mucous secretion.Furthermore, various cells including macrophages (12), neutrophils (13), eosinophils(14), and platelets (15) are capable of stimulating mast cells to release histamine.1.2.1.2. EosinophilsAn increase in the number of eosinophils in the blood, sputum, and airways ofasthmatics has been well documented (16). Although the presence of eosinophils inasthmatic airways is clear, their function has not been determined. These cells havebeen observed in the airways of patients dying in status asthmaticus (4) and have3been shown to be present in bronchial biopsy material (17) as well as BAL fluidobtained from patients with mild asthma (18). Eosinophils can be recruited into thelung by a large number of factors, and allergen challenge can increase the numbers ofeosinophils in BAL fluid of asthmatics (19). Eosinophil chemotactic factor ofanaphylaxis (ECF-A) is a family of acidic peptides with molecular weights rangingfrom 360 to greater than 1,000 that are present in the preformed granules in mast cells(20). At the lung, eosinophil secretion or phagocytosis may be stimulated viareceptors for complement and immunoglobulin. Eosinophil granules contain majorbasic protein that causes desquamation in guinea pigs (21) and has been found at sitesof damaged bronchial epithelium in asthmatic patients (22). Thus, eosinophilproducts may contribute to the respiratory tissue damage seen in asthmatic subjects.The principle lipid mediators released from stimulated eosinophils are LTC 4and LTD4 that have bronchoconstricting effects on airway smooth muscle and 15-HETE which stimulates histamine release from mast cells. Despite suggestions thatthe eosinophil is an inflammatory cell, its role as a regulatory cell in asthma has beendemonstrated due to its capability to inactivate a number of mediators. These data,along with other evidence suggest that the eosinophil has a regulatory role in asthma,although controversy exists as to the eosinophil's definitive role.1.2.1.3. MacrophagesIn the lung, the alveolar macrophages are the first cells to encounter inhaledparticles. These cells secrete numerous enzymes and mediators that initiate andregulate inflammatory reactions (23). Alveolar macrophages from asthmatic subjectshave been shown to release lysosomal enzymes by an IgE-dependent mechanism inresponse to antigenic challenge (24) and IgE-Fc receptors have been found onalveolar macrophages (25). The accessibility of this cell to antigen and its role as theprimary antigen presenting cell suggest that the macrophage may control4immunological activities in asthmatics. The addition of stimulated macrophages todog bronchial ring preparations enhances the contractile response to electrical fieldstimulation without directly causing contraction (26). This priming effect ofmacrophages correlated with the release of thromboxane A2 (TXA2), suggesting thatthis cell type enhances cholinergic nerve transmission in the airway by TXA 2 release.A wide range of products are secreted from the macrophage that are potentiallyimportant in asthma. These products include PGF 2a, PGD2, LTB4, 5-HETE, 15-HETE, and PAF (22). Although the macrophage is a potentially important cell typein the pathogenesis of asthma, the biological relevance of these findings and adefinition of its role remains to be established.1.2.1.4. PlateletsThe presence of platelet aggregation in pulmonary vessels of patients withacute respiratory failure (27) and sudden death (28) provide evidence to implicateplatelets in airway disease. Platelet aggregation appears to be altered in thecirculation of asthmatics and spontaneous platelet aggregation has been observed(29). The thrombocytopenia that occurs in allergic asthmatics challenged withantigen aerosol may cause sequestration and stimulation of platelets in the airways.Platelet factor 4 release after antigen challenge has been shown to be associated witha fall in forced expiratory volume (FEV i) suggesting that platelets have a direct rolein antigen-induced bronchospasm. Platelet-activating factor has also been shown toinduce bronchoconstriction and thrombocytopenia in guinea pigs that can bediminished by platelet depletion (30). PAF is released by various cell types includingmast cells, basophils, monocytes, macrophages, neutrophils, and platelets. Plateletsmay also be activated in the airways by ADP, epinephrine, TXA 2, serotonin,thrombin, trypsin, factor VII, immune complexes, endotoxin, viruses, and collagen(31) causing platelet aggregation in the airways. Platelets are capable of releasing5TXA2 when stimulated that has potent effects on airway smooth muscle contractionand enhances cholinergic nerve transmission in the airway (32). Although theplatelet has a central role in hemostasis, data suggests that these cells respondabnormally in the lungs of asthmatics and may be important in asthma and airwayhyperresponsiveness.1.2.1.5. LymphocytesLymphocytes are key participants in immune responses and host defense ofthe lung. In allergic asthma, B lymphocytes are committed to the synthesis ofantigen-specific IgE that is regulated by T cells. Activated T lymphocytes have beenidentified by immunohistochemistry in bronchial biopsies obtained by fiber opticbronchoscopy of asthmatic subjects (33). An increase in the proportion of circulatingT lymphocytes that are activated from patients with severe acute asthma has beendemonstrated by enhanced antigen expression (34). Decreased antigen expression ofthese activation markers may be attributed to corticosteroid treatment during asthmaexacerbations (34). Individual T cell clones can provide support for IgE productionmediated by interleukin 4 (35) as well as releasing lymphokines which attract andactivate neutrophils (36). Also, the activation of TH-lymphocytes is associated withthe expression of mRNA for IL-2,3,4,5,GM-CSF, and IFNy in asthmatic BAL cellspossibly mediating allergic asthma (37). Increased natural killer activity has beendescribed in the peripheral blood of asthmatic patients and is a nonspecific indicatorof lymphocyte activation (38). T cells have the capacity to produce a wide variety ofcytokines relevant to the inflammation seen in asthma, although their precise role inthe development of inflammatory responses to inhaled antigen is uncertain.61.2.1.6. NeutrophilsExperimentally induced airway hyperresponsiveness in laboratory animals isassociated with an inflammatory cell influx into the airways consisting primarily ofneutrophils (39). The development of hyperresponsiveness in dogs can be preventedby granulocyte depletion with hydroxyurea (40), and the number of circulatinggranulocytes has been shown to be associated with the degree of airwayhyperresponsiveness (41) implicating an important role for these cells. Although theinflammatory cell infiltrate consists initially of neutrophils and subsequentlyeosinophils, the development of airway hyperresponsiveness correlates with theneutrophil influx (42). The role of inflammation in antigen-induced bronchospasmand airway hyperresponsiveness have been investigated by antigenic challenge ofimmunized animals.The influx of neutrophils observed in postmortum tissue, bronchial biopsy,and BAL implicate the neutrophils in asthma and airway hyperresponsiveness. Usinggranulocyte depletion studies in rabbits sensitized with serum containing antiragweedIgE, the neutrophil influx into the airway, the late asthmatic response, andhyperresponsiveness to inhaled histamine were prevented, but reconstituted withneutrophil repletion (43). These results suggest that there is a requirement forneutrophils at the time of antigen challenge to mediate airway responses. Themechanism of neutrophil recruitment, the stimuli causing movement into the airway,and the products of the neutrophil in the airway that are involved in asthma are notwell defined. These cells may be recruited into the airways by a broad range ofagents. Chemoattractant receptors present on neutrophils include the receptors forformylated peptides derived from bacterial cells, leukotriene B4 (LTB 4), platelet-activating factor (PAF), and small complement fragment C5a. Numerous otherreceptors are present on the surface of neutrophils that control functions such asadherance and phagocytosis using the leukocyte adhesion molecule (LEUCAM)7proteins and immunoglobulin receptors, respectively. As well as stimulatingneutrophil chemotaxis via regulating actin polymerization, chemotactic factors primeneutrophils for phagocytosis and microbiocidal activities. In the airway, neutrophilsare the source of a number of lipoxygenase and cyclooxygenase lipid products thatare capable of contracting smooth muscle. Evidence suggests that the neutrophilplays an important role in inflammation and hyperresponsiveness in asthma.1.2.2. Effector cells1.2.2.1. Epithelial cellsAirway epithelial cells are located in an ideal position to interact withenvironmental conditions and modulate the responses of other airway cells. Thesecells may function in part by regulating smooth muscle responsiveness (44) as well asreleasing chemotactic factors for inflammatory cells including neutrophils,monocytes and lymphocytes (45). Destruction of the epithelium is present at alllevels of the airways in asthmatic patients caused by airway inflammation (17).Airway epithelial damage may expose unmyelinated C-fiber nerve endings, which arestimulated by inflammatory mediators such as bradykinin. C-fibers may containsensory neuropeptides such as substance P that may cause an axon reflex andtransient bronchoconstriction (46). As well as easier access of irritant factors to nerveendings, epithelial damage may contribute to bronchial hyperresponsiveness byenhancing penetration of allergen particles to mediator secreting cells. Overall,epithelial disruption and shedding are important features of asthmatic airwaysresulting from the activation of inflammatory cells.1.2.2.2. Smooth muscle cellsAirway smooth muscle is found along the entire length of the tracheoalveolartree. In the trachea, the airway passage is surrounded by a series of C-shaped hyaline8cartilages that function to keep the trachea from collapsing. The trachealis smoothmuscle joins the cartilage posteriorly forming the tubular trachea. The walls of theprimary bronchi are also partially supported by cartilaginous rings, although smoothmuscle encircles the bronchus. In further branches of the airways, the cartilagebecomes fewer and smaller, and the smooth muscle surrounding the airways becomesmore prevalent. The airways of the bronchioles contain no cartilage and arecompletely surrounded by smooth muscle that is responsible for asthmatic symptoms.During an asthmatic attack, the bronchial muscles undergo spasms and passagewaysare easily constricted without cartilaginous support. In addition, a major source ofresistance to airflow and the site of the largest pressure drop is in the more centralairways (47). Therefore, upper airway narrowing due to trachealis contraction as wellas contraction in the lower airways combine to cause breathing difficulty duringasthmatic attacks. Control of airway smooth muscle contraction is likely responsiveto neural and myogenic control systems that can be modified by inflammatorystimuli. These cells are the end-effector cells that contribute to obstructive airwaydisease. Activation of airway smooth muscle involves complex multicellularinteractions that have not yet been defined.Airway hyperresponsiveness is an increased contractile reactivity of airwaysmooth muscle and is considered to be a characteristic feature of current,symptomatic asthma. Inhalation of a number of stimuli such as allergens (48), ozone(49) and low molecular weight chemical sensitizers, such as toluene diisocyanate (50)or plicatic acid (51) can cause hyperresponsiveness in human subjects. Thus,commonly encountered environmental allergens are associated with persistingsymptoms and allergen avoidance has been shown to improve both airwayhyperresponsiveness and asthmatic symptoms (52). Stimili that cause airwayhyperresponsiveness in humans also cause similar changes in animal models thathave been developed to investigate the pathogenesis of airway hyperresponsiveness.9Mammalian airway smooth muscle contraction decreases airway diameter andincreases resistance to airflow. A variety of extracellular signals including hormones,neurotransmitters, drugs, and mediators control smooth muscle tone. Specific surfacereceptors on airway smooth muscle cells are activated by ligands and transduced tostimulate contractile proteins. P-adrenoceptors, a-adrenoceptors, cholinergicreceptors, neuropeptide receptors, and inflammatory mediator receptors are presenton airway smooth muscle as detected by functional studies and direct bindinganalysis (53). Airway smooth muscle contraction in response to inflammatory cellmediator release causes airway narrowing, decreased airflow, and shortness of breath.A better understanding of the signal transduction machanisms leading to smoothmuscle contraction may lead to the development of therapy for asthma andcardiovascular diseases.1.3. CHEMICAL MEDIATORS IN ASTHMA.Release of inflammatory mediators from primary effector cells forms acomplex process that likely result in airway narrowing and hyperresponsiveness (54).The most characteristic feature of asthma is the increased bronchial reactivity to awide variety of pharmacological and physiolgical agents. Advances in biochemistryand pharmacology have led to better assays for detecting mediators and developingantagonists to investigate their roles in asthma. Inflammatory mediators may begenerated by resident cells within the airways and lung, or by cells that have migratedto the lung from circulation. Histamine was one of the first mediators identified inasthma, although more recently the cyclooxygenase and lipoxygenase products ofarachidonic acid metabolism have been active areas of investigation. Sensoryneuropeptides such as substance P are released from airway nerves have also beenlinked to asthma due to noncholinergic neural mechanisms of smooth muscle10contraction and increased mucous secretion (55). Although some mediators arecapable of reproducing some of the pathological changes associated with asthma, it islikely that many mediators are involved. These mediators link inflammatory cellactivation with the airway responses of both epithelial and smooth muscle cells.Muscle spasm is caused by histamine interactions with its H I-receptor, as wellas contributions by leukotrienes C, D, and E, prostaglandins, thromboxanes,bradykinin and platelet-activating factor (PAF). Acetylcholine and neuropeptidesalso have a secondary effect although the cumulative effects of multiple mediators isthought to induce muscle spasm. Mucosal edema as a result of increased vascularpermeability is caused by histamine, leukotrienes, prostaglandins, bradykinin, andPAF. Chemotactic factors, leukotriene B4, and HETES stimulate cellular infiltrationfrom circulation into the airways. Leukotrienes are the most potent stimulus formucous secretion, followed by HETES, whereas histamine is the least active. Duringinflammation, however, 5- and 15-HETES production is increased dramatically,implicating an important role for HETES. Also, arachidonic acid and PAF stimulatethe release of 15-HETE from human epithelial cells that is capable of contractingbronchial smooth muscle (56).Bronchoconstriction in vivo to inhaled histamine suggests that this mediatorplays an important role in allergically mediated airway disease. A variety of inhaledmediators are capable of causing reversible hyperresponsiveness. Prostaglandin F 2a(PGF2a) (57), PGE 1 and PGE2 (58), PAF (59), LTC4 and LTD4 (60), histamine (61)as well as TXA2 (62) increase the responsiveness of airway smooth muscles. Thesemediators may also promote mucosal edema and glandular secretion, causing airwaynarrowing and limiting airflow. However, the mechanism of airway constriction isnot clear, and may result from the direct effect of these mediators or their effect onother cells that produce contractile agonists. Although PAF is a potentbronchoconstrictor in humans (58), it has no direct effect on smooth muscle in vivo.1 1Therefore, PAF-induced bronchoconstriction is an indirect result of activating PAFreceptors present on platelets (63), neutrophils (64), macrophages (65), lung tissue(66) and I have demonstrated PAF binding to human lymphocytes (P.J.D. and H.Salari, unpublished observations). Since mast cells are capable of synthesizingLTC4 , LTD4 and histamine, release of these substances from stimulated mast cellsmay induce smooth muscle cell contraction. Discrete receptors for LTC4 and LTD4have been identified in guinea pig trachea (67). LTC 4 and LTD4 stimulatepolyphosphoinositide hydrolysis in guinea pig trachea and inhibit adenylate cyclaseactivity leading to contraction (68). Airway epithelial cells also produce 5-HETE and15-lipoxygenase products such as 8,15-diHETE that have chemotactic properties forneutrophils (69).The intercellular effects of inflammatory mediators are of interest incontrolling both airway smooth muscle and inflammatory cells. The release andgeneration of several inflammatory mediators may be enhanced in asthma. In BALfluid, an increase in histamine (18) and PAF (70) have been identified in asthmaticsubjects compared with a normal population. A priming effect of several mediators isa potential mechanism for the enhanced responses in eosinophil, neutrophil, andsmooth muscle cell activation.Since a large number of mediators may be involved in asthma, it is unlikelythat one particular mediator is responsible for all the pathophysiological aspects ofasthma. The major problem with understanding the function of individual mediatorsis that many of them function simultaneously and produce interactive results (71).Therefore, antiasthmatic therapy using antagonists has not been an acceptable form oftherapy. Accordingly, anti-histamines have been less effective than general anti-inflammatory agents such as corticosteroids, which act at several sites to preventmediator release and suppress the effects of released mediators. /3-adrenoreceptoragonists stimulate adenylate cyclase and cAMP formation by acting on various cells12including airway smooth muscle cells and leukocytes causing bronchial smoothmuscle relaxation and inhibition of inflammatory mediator release, respectively.Xanthines such as theophylline are less potent than 13-agonists with similar effects,relaxing smooth muscle and decreasing mediator release. Antiallergic drugs such asnedocromil sodium, ketotifen, and histamine H 1 -receptor antagonists have mast cellstabilizing effects but are only useful against antigen-induced responses (72).Prednisone and prednisolone are the most widely used orally administeredcorticosteroids which stimulate the mRNA transcription of lipocortins that inhibitphospholipase A2 (73), an enzyme responsible for the production of prostaglandins,leukotrienes, and PAF. Although several medications are available to relieveasthmatic symptoms, there still exists significant limitations in the control ofasthmatic reactions, particularly bronchial hyperresponsiveness.1.4. ACTIVATION AND SIGNAL TRANSDUCTION IN NEUTROPHILSTissue inflammation is associated with the infiltration of leukocytes. Thesecells adhere to the blood vessels and migrate between interendothelial junctionsmoving towards the site of inflammation. This process of directional migration iscalled chemotaxis. In early stages of the inflammatory response, the predominant celltype is the polymorphonuclear leukocyte, or neutrophil. These cells respond at thesite of inflammation by ingesting and disposing of unwanted material, such asbacteria or broken down cells. Inside the phagocytic vacuole, these terminallydifferentiated phagocytic cells use granule contents and reactive oxygen species asmicrobiocidal agents.When activated, neutrophils accumulate in alveolar blood vessels and play afundamental role in initiating acute tissue injury. Neutrophils are rich in granules andare able to synthesize low amounts of proteins. These motile cells are able to13selectively release stored granule contents and can generate reduced oxygen speciesby a unique enzyme, NADPH-oxidase. In vitro studies have been used to determinethat stimulated neutrophils are capable of inducing morphologic and functionalchanges in the pulmonary endothelium (77). Changes in the pulmonary endotheliumas a result of neutrophil activation include: the disruption of endothelial cell plasmamembrane, hemorrhage and intravascular coagulation, the generation of lipidperoxidation products, and the development of protein-rich pulmonary edema.Although scavengers of reactive oxygen species are capable of inhibiting some ofthese changes, proteases are likely to contribute to endothelial cell cytolysis (78).Therefore, the observed endothelial cell injury is a result of the synergistic effect ofoxygen metabolites and lysosomal proteases released from activated neutrophils.Leukocyte chemotaxis was first demonstrated in 1962 by Boyden's in vitroassay system capable of detecting migration of leukocytes towards serum treatedantigen-antibody complexes (79). Migration from circulation to sites ofinflammation is dependent on the ability of phagocytes to detect factors produced atareas of inflammation or factors produced by invading microorganisms (80). Bothpeptide and lipid materials are capable of inducing potent chemotactic activities inneutrophils. N-formylpeptides are by-products of bacterial protein synthesis thathave been shown to bind to neutrophil cell surface receptors using radiolabeledFMLP. Various N-formylpeptides inhibit fMet-Leu-[ 311]Phe binding, suggesting thata common receptor is used by different formylated peptides to induce biologicalresponses (81). Binding characteristics of FMLP to whole human neutrophils suggestthe presence of approximately 50,000 receptor binding sites per cell and a Kd of -20nM (81). Analysis of binding to membrane preparations, however, indicates that highand low affinity receptors are present with approximately 75% being of the lowaffinity class (81). The FMLP receptor that has been purified appears to be heavilyglycosylated resulting in a decreased molecular size after removal of carbohydrates14(82). The FMLP receptor consists of several glycoprotein components ranging in sizefrom 50-70 kDa, and deglycosylation reveals a core polypeptide of 32 kDa that is stillable to bind the ligand (82). Using a cDNA library created from HL-60 cellsdifferentiated into granulocytes highly responsive to FMLP, a 1.9 kb open readingframe encoding a 350 residues protein was isolated (83). The hydrophobicity plotindicates that this receptor has seven transmembrane domains and is an example of aG-protein coupled receptor with two potential N-linked glycosylation sites.During the formation of antigen-antibody cascades, serum complementprotein components C3a and C5a induce neutrophil chemotaxis and histaminereleasing activity (84). These cells have specific receptors for complement peptideC5a with an estimated 50,000 to 113,000 receptors per cell and Kd -2 nM (85). Likethe FMLP receptor, this receptor also appears to have a high and low affinity siteswhen binding to membrane preparations is analyzed. The signal transductionpathway of the C5a receptor became clearer when a 42 kDa C5a receptor wascopurified as a tightly coupled complex associated with pertussis toxin-sensitive G-protein a and 13 subunits (86). A high affinity C5a receptor has been cloned and thededuced amino acid sequence reveals a receptor with Mr of 39,000 with sevenhydrophobic transmembrane spanning motifs, an N-linked glycosylation site, andputative G-protein interaction sites (87).After activation, neutrophils generate several arachidonic acid metabolitesincluding PGE2, TXA2, 5-HETE, and LTB4. Of the various arachidonic acidmetabolites, LTB4 is the most potent chemotactic factor for neutrophils andeosinophils known to date. LTB 4 is also a potent proinflammatory agent that causesa number of responses in leukocytes including chemotaxis, lysosomal degranulation,oxygen radical generation, and an increase in leukocyte adhesiveness (88). In tissues,LTB4 causes edema and the wheal and flare reaction. The LTB 4 receptor inneutrophils is thought to be coupled to an inhibitory G-protein (Gi), since pertussis15toxin inhibits LTB4-induced responses such as the mobilization of intracellularcalcium, stimulation of phosphatidylinositol turnover, pH changes, and degranulation(89, 90). This lipid serves as a stimulus for neutrophil chemotaxis by binding tomembrane receptors that may be present in two affinity states. High affinityreceptors with a Kd -0.12 nM, -4,400 sites and low affinity receptors with a Kd -50nM, -270,000 sites are reported that mediate neutrophils responses to LTB 4 (91).In addition to molecular cloning, covalent cross-linking studies haveidentified protein receptors with molecular masses ranging from -50-70 kDa usingradiolabeled FMLP, C5a, and an LTB 4 analogue. These receptors also appear to beassociated with G-proteins by analysis of binding in the presence of GTP analogues.As well as their role in signal transduction, these G-proteins are though to beinvolved in regulating the interconversion between high and low affinity receptorspresent on neutrophil cell surfaces. The LTB 4 receptor will likely soon be cloned,since cell lines such as WEHI-3B (P.J.D. and H. Salari, unpublished observations)have been identifed that have a LTB 4 receptor and methods to clone receptor for lipidmediators have been established (92).Another lipid mediator that is released by neutrophils and acts on bothplatelets and neutrophils is platelet-activating factor (PAF). This ether-linkedphospholipid is released from various cell types and binds specific receptors presenton human neutrophil membranes. Some groups have reported the presence of asingle class of receptor for PAF with a Kd of 0.57 nM (93) while other investigatorsbelieve high affinity, Kd 0.2 nM and low affinity, Kd 500 nM receptor sites exist(64). Guanine nucleotides affect PAF binding suggesting G-protein involvement inthe signal transduction of PAF (93). However, the class of G-protein that interactswith the PAF receptor is unknown. Insights into the signal transduction pathwayresulting from PAF receptor activation were achieved by cloning a -39 kDa PAF16P PI P3Ca2+LYSOSO MA LENZYMESNAD PHDASE02 	02H202p, 	 D AGPI P2receptor from guinea pig lung having seven transmembrane domains and potential G-protein interaction sites (92).Extracellular signaling molecules (ligands) bind to receptors present on targetcells. Ligand/receptor interactions initiate a cascade of intracellular events leading tocellular activation and controlling cell functions (see Figure 1). Various componentsof signal transduction pathways have been used to model the events leading tocellular stimulation. Two classes of receptors have been implicated in the activationof separate signaling systems, however some components are common to bothsystems. Receptors that possess a protein-tyrosine kinase domain respond to agonistsby directly phosphorylating substrates as well as autophosphorylation. On the otherhand, receptors that have seven transmembrane domains are coupled to aheterotrimeric guanine nucleotide binding protein (G-protein) that is the firstcomponent activated as a result of ligand/receptor interactions.CH EMOTACT ICFACTOR17Figure 1- Model of signal transduction leading to neutrophil activation.Neutrophil responses to extracellular ligands are essential for effective hostdefenses against invading microorganisms and destruction of foreign substances.Responses resulting from the formation of receptor-ligand complexes in neutrophilsinclude chemotaxis, increased adherance, release of inflammatory mediators andlysosomal enzymes, activation of the respiratory burst, and cytoskeletalrearrangement. G-proteins have been implicated in coupling receptors to variousintracellular pathways that are stimulated by extracellular molecules and lead tochemotaxis and activation of these phagocytic cells. A number of closely relatedproteins having structural, functional, and regulatory homology make up the signaltransducing G-protein family (94). These proteins are localized on the cytoplasmicsurface of membranes and are composed of three distinct subunits, designated a, (3,and y. The a subunit is the largest subunit (Mr 39,000-54,000) that binds guaninenucleotides, hydrolyzes GTP into GDP, and plays a major role in initiatingintracellular events. The (Mr 35,000-36,000) and 7 (Mr 8,000-10,000) subunits areassociated as a complex that interacts with the a subunit that binds GDP when the G-protein is not activated. In response to stimulus, the a subunit displaces GDP forGTP and dissociates from the regulatory [37 subunits. In this activated form, the freea subunit bound to GTP interacts and activates downstream second messengersystems. As well as the receptor transducing heterotrimeric GTP-binding proteins,there is a growing group of low molecular weight monomeric ras-related proteins-20-30 kDa in size (95) that may play a role in neutrophil activation. The geneproducts of rho as well as elongation and ADP-ribosylating factors and a number ofrecently identified proteins such as Rab, Rap, and Ral are a few members in thisgroup which have homologies with the GTP-binding a subunit of the heterotrimericG-proteins (96).Downstream of G-protein activation, different pathways are activateddepending on the type of G-protein that is coupled to the cytosolic (intracellular)18portion of the receptor. In many cases, the Gp class of G-protein activates aphosphatidylinositol specific phospholipase C (PLC) that hydrolyzesphosphatidylinositol bisphosphate (PIP2) releasing water soluble inositoltrisphosphate (IP 3) and lipid associated diacylglycerol (DAG). Multiple isoforms ofPLC have been purified from mammalian tissues having similar catalytic properties,hydrolyzing inositol phospholipids. Antibodies prepared against purified PLC wereused to screen a cDNA library and clones corresponding to PLC a, (3, y, and 5 havebeen isolated. When released from the membrane, 1133 binds to 1P 3-specific receptorson intracellular Ca2+ stores causing the release of calcium into the cytosol (97).Increases in intracellular calcium activate calmodulin-dependent protein kinases andsynergizes with DAG to activate the Ca 2+-activated, phospholipid-dependent proteinkinase, protein kinase C (PKC) (98). Although neutrophil activation involves theabove signal transduction pathways, it is unclear how PKC activation is linked toneutrophil responses. Phorbol esters have been shown to stimulate superoxide anionproduction by redistributing PKC (99) although a direct effect of PKC on NADPH-oxidase is unknown. Since PKC inhibitors partially inhibit superoxide production, ithas been suggested that other signal transduction mechanisms are involved incoupling receptor activation with neutrophil responses (100).The src homology region 2 (SH2) is a noncatalytic region of -100 aminoacids conserved on a series of cytoplasmic signalling proteins (101). Activated EGF-and PDGF-receptors complex with a set of cytoplasmic proteins including PLCy,p2lras GTPase activating protein (GAP), phosphatidylinositol (PI) 3'-kinase andp74raf (102). When activated, EGF- and PDGF-receptors undergoautophosphorylation on tyrosine residues that may interact with conserved positivelycharged residues within SH2 domains (103). Association between the receptors andPLCyl redistributes PLCyl from the cytosol to the membrane where its substrate,PIP2, is located (104). Bound PLCyl is then tyrosine phosphorylated by the receptor,19leading to activation of PLC and PIP2 hydrolysis. Thus, conserved SH2 domainshave been identified on several signal transducing proteins and mediate theassociation, translocation, and activation of downstream signal transduction fromactivated receptor tyrosine kinases.Mitogen-activated protein (MAP) kinases are serine/threonine kinases that areactivated by a variey of hormone and growth factors (105). This family of proteinkinase is active when phosphorylated on both serine/threonine and tyrosine residuesproviding a link between protein-tyrosine and protein-serine/threonine kinases (106).One of these MAP kinases is a 42 kDa protein that has been detected onantiphosphotyrosine immunoblots in fractions exhibiting MAP kinase activity. Arecent report demonstrates that MAP kinases are tyrosine phosphorylated andactivated in neutrophils treated with GM-CSF, interleukin-3, and Steel factor (107).The role of these kinases in neutrophils may help clarify unknown mechanismsleading to neutrophil activation. MAP kinases are thought to be located at a uniqueposition in signal transduction mechanisms and may be responsible for integratingsignals from pathways involving different classes of protein kinases. The linkbetween early activation of the biochemical pathways discussed above andphysiological cell responses is unclear. In this report, protein-tyrosine kinaseinhibitors were used and their effects on cellular activation have been analyzed inneutrophils and smooth muscle cells.1.5. PROTEIN-TYROSINE KINASE ACTIVITY IN NEUTROPHILSPhosphorylation on tyrosine residues by protein kinases was first identified invirally-transformed cells. Several genes encoding protein-tyrosine kinase activityhave now been cloned that share sequence homology over a stretch of approximately300 amino acids defined as the catalytic domain (108). In addition, these kinases20share common features with serine/threonine kinases in both the catalytic domain andthe highly conserved ATP-binding Gly-X-Gly-X-X-Gly-X 15_20-Lys motif (109). Alarge proportion of oncogene-encode protein-tyrosine kinases are activated intransformed cells. An alteration in signal transduction normally controlled by growthfactor stimulation of protein-tyrosine kinases leads to the uncontrolled growth ofneoplastic cells. Although tumor cells were initially found to contain elevatedprotein-tyrosine kinase activity, these enzymes have been shown to be components ofnormal cellular functions. Many growth factors and cytokines are activators ofprotein-tyrosine kinases in target cells that control cell differentiation and/orproliferation.Gomez-Cambronero et al. (110) reported protein-tyrosine phosphorylation offive proteins with molecular masses of 118, 92, 78, 54, and 40 kDa in neutrophilsstimulated with granulocyte-macrophage colony stimulating factor (GM-CSF).Cytosolic and particulate substrates have been shown to be tyrosine phosphorylatedin neutrophils stimulated with FMLP, LTB 4 , a phorbol ester, and calcium ionophoresuggesting receptor-dependent and receptor-independent protein-tyrosine kinaseactivation (111). Proteins with similar molecular weights have also been shown to betyrosine phosphorylated in neutrophils stimulated with the chemotactic peptideFMLP (112). In addition, similarities in the patterns of protein-tyrosinephosphorylation are also evident in neutrophils activated with PAF and LTB 4 (113,114). Attempts have been made to identify these phosphoproteins and to characterizetheir roles in neutrophil activation. In contrast to some growth factors such as EGF,PDGF, c-kit ligand and insulin (109), the receptors for inflammatory agonists such asPAF, LTB4 , and FMLP have not been shown to have a kinase domain. In fact, thePAF (92) and FMLP (83) receptors that have been recently cloned appear to be linkedto G-proteins but no indication of how a protein-tyrosine kinase might be activated.This suggests that the stimulation of protein-tyrosine kinases by receptors for21inflammatory mediators is occuring as a consequence of other second messengersystems downstream of G-proteins.Stimulation of protein-tyrosine phosphorylation in electroporated humanneutrophils treated with GTPyS suggests that the activated G-proteins can stimulateprotein-tyrosine kinases (115). Similarily, receptor-mediated protein-tyrosinephosphorylation has been shown to be mediated in part by a pertussis-sensitive G-protein (110). Various studies have demonstrated tyrosine phosphorylation of PLCyfollowing interaction with ligand-activated PDGF of EGF receptors (116-118). PLCa, 13, and 8 are activated by receptors coupled to a G-proteins, however, PLCyactivity increases following tyrosine phosphorylation (119). Therefore, the preciserelationship between protein-tyrosine kinase activation and activation via G-proteinsremains unclear.1.6. PROTEIN KINASES IN SMOOTH MUSCLE CELLSThe major protein components of the contractile apparatus of airway smoothmuscle include actin, myosin, tropomyosin, caldesmon, calponin, myosin light chainkinase (MLCK), and protein phosphatases (120). MLCK activity is controlled by[Ca21i and by phosphorylation state as regulated by other protein kinases (121) orprotein phosphatases (122). Phosphorylation of MLCK may have an important rolein cross-bridge regulation, myosin light chain phosphorylation, and mechanicalresponses of smooth muscle to mediators of airway constriction. There are severalprotein kinases activated by mediators of airway smooth muscle contraction includingcAMP- and cGMP-dependent protein kinases, protein kinase C (PKC), and themultifunctional Ca2+/calmodulin dependent protein kinase (CaM kinase II). cAMP-dependent protein kinase (PKA), PKC, and CaM kinase II all phosphorylate MLCKat the regulatory site, although only PKA and CaM kinase II do so at a rate sufficient22to have a role in regulation (121). Also, it appears that agonists do not activate PKCsufficiently to phosphorylate myosin light chains directly and do not induce anychanges in heavy chain phosphorylation (123). Although the above serine/threoninekinases have been investigated using kinase inhibitors, activators, and in vitro kinaseassays, protein-tyrosine kinase activity has yet to be investigated in smooth musclecells.Extracellular ligands acting on airway smooth muscle cells tranduce signalsresulting in increasing intracellular calcium levels. Two mechanisms of increased[Ca21i are voltage dependent influx of extracellular Ca2+ and IP 3-induced Ca2+release from intracellular stores. Although myoplasmic calcium has been proposed tobe an important component of the contractile process, no simple relationship exists tolink [Ca2-l i with force and the mechanism of return to basal [Ca 2-1i and relaxation(76). Some models of smooth muscle contraction suggest that MLCK is activated by[Ca21i resulting in phosphorylation of myosin light chains. Controversy exists,however, since Ca2+ depleted tissue does not support contraction when myosinphosphorylation occurs suggesting that [Ca2-l i is of prime importance (124).As well as smooth muscle contraction, models of airway narrowing suggestthat increasing the amount of smooth muscle contributes to increased airflowresistance (125). Therefore, Type 13 PDGF receptors detected on bovine smoothmuscle cells (126) suggests that protein-tyrosine kinase activity may play a role inairway smooth muscle proliferation associated with asthma. In a recent study,smooth muscle contraction in guinea pig taenia coli was observed in response tovanadate, a protein-tyrosine phosphatase inhibitor (127). Therefore, studying thepresence of protein-tyrosine kinase activity and its contribution to airway smoothmuscle cell contraction is an appealling investigation.231.7. PROTEIN-TYROSINE KINASE INHIBITORSErbstatin was the first inhibitor of protein-tyrosine kinase activity isolatedfrom the culture filtrate of Streptomyces sp. (128) that competes with the peptidesubstrate (129). Analogs of erbstatin have since been synthesized (130, 131) in anattempt to produce selective, non-toxic, and potent inhibitors of protein-tyrosinekinases. Low molecular weight protein-tyrosine kinase inhibitors that compete forthe substrate binding sites of protein-tyrosine kinases have been termed tyrphostins.As well as inhibitors of tyrosine-containing substrates such as erbstatin, novelapproaches have been used to made to synthesize inhibitors of protein-tyrosine kinaseactivity that mimic the enzymes' transition state (132) and the nucleoside triphosphatebinding site (133).Protein-tyrosine kinases are a potential target for drug design. The phenolicresidue of tyrosine is a distinctive structure that receives a phosphate in a well definedchemical reaction catalyzed by a protein-tyrosine kinase. On the other hand, ATPantagonists such as genistein and quercetin are likely to be much less selectivechemical agents and inhibit the activities of other protein kinases. This undesirablefeature may cause cytotoxicity and decreased selectivity. Therefore, tyrphostinscontaining phenolic structures that are slightly hydrophobic to cross membranes andlow molecular weight non-peptide compounds to enhance biological stability havebeen the most effective protein-tyrosine kinase inhibitors (134). With thesecharacteristics, future drugs may be designed that are capable of achieving selectivetherapy with minimal side effects. These synthetic compounds may have applicationsin treating specific pathological conditions involving protein-tyrosine kinaseactivities. For example, psoriasis can be treated with tyrphostins since theoverexpression of transforming growth factor a enhances EGF receptor activity andepidermal hyperplasia of keratinocytes (135).24Protein-tyrosine kinases catalyze the phosphorylation of tyrosine onexogenous substrates, therefore synthetic copolymers of amino acids containingtyrosine have been used. Small peptides such as angiotensin and the src peptide havebeen used as substrates, although the optimal substrate for some protein-tyrosinekinases have been shown to be polymers containing 80% glutamic acid 20% tyrosine(136). Analysis of protein-tyrosine kinase inhibitors have used several enzymesources including purified enzymes, partially purified enzymes, immunoprecipitatesfrom broken cells, or cell extracts. Assays for EGF receptor kinase (131, 137) andinsulin receptor kinase (138) have been commonly used to analyze the potency ofprotein-tyrosine kinase inhibitors.Previously described protein-tyrosine kinase inhibitors were used in this studyas probes to analyze protein-tyrosine kinase activity in neutrophil and smooth musclecells. These compounds are derived from the benzylidenemalononitrile nucleus andresemble both tyrosine and erbstatin. The benzylidenemalononitriles are easy toprepare and are stable in comparison with other erbstatin derivatives (131). It hasbeen possible to synthesize benzylydene compounds with a 2940-fold increase inEGF receptor kinase affinity through specific substitutions (137). a-Cyano-3,4-dihydroxythiocinnamamide (DHC, AG 213, tyrphostin A47, RG-50864, Figure 2)was synthesized by Knoevenagel condensation of cyanothioacetamide (139) and wasfound to be a more potent inhibitor of the EGF receptor kinase than the insulinreceptor kinase (137). Using a synthetic polymer containing tyrosine as a substrate,the EGF receptor kinase was competitively inhibited by DHC with an IC 50 of 2.4 pMand a Ki of 0.85 tM (131). This compound was found to be a poor inhibitor ofserine/threonine kinases with IC 50 values of >100 p.M and 65 gM for PKC andcAMP-dependent protein kinase, respectively. Although recently DHC has beenmade commercially available, DHC was not available for analysis of smooth musclecontraction. Therefore, another previously characterized protein-tyrosine kinase25inhibitor, 4-hydroxy-3,5-diisopropylcinnamamide (DIPC, ST271), was used to studyairway smooth muscle contraction. DIPC was also a potent EGF receptor kinaseinhibitor with an IC 50 of 1.1 p.M using plasma membrane fractions as an enzymesource (140). Phosphoamino acid analysis of DIPC treated cells confirmed that DIPCdecreased the incorporation of phosphate into tyrosine residues without affectingphosphorylation of either serine or threonine (140, 141). DIPC also inhibited pp60e -src in a dose-dependent manner with an IC 50 of 4.4 RM. Thus, the protein-tyrosinekinase inhibitors used in this study are suitable for investigating the role and functionof protein-tyrosine kinases in neutrophils and airway smooth muscle cells.S26HOHOiso-PrHOiso Pra -cyano-3,4-dihydroxythiocinnamamide 	 4-hydroxy-3,5-diisopropylcinnamamide(DHC) 	 (DIPC)Figure 2 - Structures of protein-tyrosine kinase inhibitors.Although protein-tyrosine kinases have been primarily associated with growthfactors associated with the onset of oncogenesis, tyrphostins have also been used toassess protein-tyrosine kinase activity in normal cellular functions. Cellularproliferation dependent on protein-tyrosine kinase activity can be blocked bytyrphostins (137, 138). Platelet aggregation and activation in response to PAF andthrombin can be inhibited by both erbstatin (142) and an ATP binding sitecompetitor, genistein (143). The effect of erbstatin was also investigated inneutrophils and found to have a partial effect on neutrophil activation at highconcentrations (144). Also, B lymphocyte IgM-induced proliferation, oncogeneexpression, protein-tyrosine phosphorylation, and increases in [Ca2+] i, IP and IP3levels were blocked by tyrphostins (145). Therefore, inhibitors of protein-tyrosinekinase activity have been useful probes to assess the roles of protein-tyrosine kinasesin cell activation and function.1.8 OBJECTIVESInflammation and hyperresponsiveness are characteristic features seen inasthmatic patients that involve neutrophils and smooth muscle cells, respectively.Based on experimental evidence, Nadel and Holtzman (74) proposed a mechanismfor smooth muscle hyperresponsiveness involving neutrophil activation (see Figure3). In the airway, inflammatory stimuli interact initially with epithelial cells,stimulating the generation and release of lipid chemotactic factors including LTB 4.These factors induce infiltration of neutrophils from the microcirculation into theairway epithelium where they become activated. Cyclooxygenase and lipoxygenaseproducts may then be generated and released from neutrophils that may act onsmooth muscle cells and the nerves that regulate contractile responsiveness. Usingthis model, the present study was carried out to gain a more complete understandingof neutrophil and smooth muscle cell activation.27INFLAMMATORYSTIMULUS28I 	 IEPITHELIUM1 /NEUTROPHIL \OACT/VAT/ON/LT84 	 NEUTROPHILRELEASE 	 CHEMOTAXISHYPERRESPONSIVENESS  O O0CAPILLARYFigure 3 - Possible mechanism of airway smooth muscle hyperresponsiveness. Seetext for description. (From ref. 74)In neutrophils, the role of protein-tyrosine phosphorylation in response toagonist stimulation has not been clearly defined. Protein-tyrosine kinase activitiesfrom cytosolic and particulate neutrophil fractions have been partially characterized(75) and it has been reported that the activation of neutrophils by various agonistsinvolves phosphorylation on tyrosine residues. These investigations suggest thatprotein-tyrosine phosphorylation plays a role in neutrophil stimulation. The proposedmechanism of smooth muscle cell signal transduction leading to contraction does notinvolve protein-tyrosine phosphorylation (76). Using protein-tyrosine kinaseinhibitors, the role of these kinases were probed in neutrophils and smooth musclecells.My specific aims were: 1) to analyze the role of protein-tyrosine kinaseactivity in neutrophil activation using a protein-tyrosine kinase inhibitor; 2) todetermine the site of action of protein-tyrosine kinase inhibitors in the signaltransduction pathway leading to neutrophil activation; and 3) to characterize andpartially purify protein-tyrosine kinase activity from airway smooth muscle cells.These studies will provide new insights into the role of protein-tyrosine kinaseactivity in neutrophil activation and the potential use of these drugs asantiinflammatory agents. The identification of protein-tyrosine kinase activity insmooth muscle cells provides a new site of intervention for diseases resulting fromsmooth muscle contraction. Ultimately, knowledge of the signal transductionmechanisms that are common to several inflammatory mediators in both neutrophilsand smooth muscle cells should have potential application in the management ofasthma.29CHAPTER 2MATERIALS AND METHODS2.1. MATERIALSHistone H1, BSA, ovalbumin, L-a-phosphatidyl-L-serine, DAG,phenylmethyl sulfonyl fluoride (PMSF), leupeptin, pepstatin, P-methylaspartate,diisopropylfluorophosphate (DFP), pepstatin, LTB4 , PAF, FMLP, histamine,carbamylcholine chloride (carbachol), collagenase, elastase, Ficoll-Hypaque,cytochalasin B, cAMP-dependent protein kinase inhibitor peptide (amino acids 5-24)(PKIP), non-enzymatic cell dissociation buffer, and other chemicals, unless stated,were purchased from Sigma Chemical Co. (St. Louis, Mo). LTD 4 was purchasedfrom Cayman Chemical Co (Ann Arbor, MI). Fura-2AM was purchased fromMolecular Probes (Eugene, OR). Dextran T500, FPLC pumps, and FPLC columnswere from Pharmacia (Montreal, Canada). Electrophoresis reagents, nitrocellulosesheets, and alkaline phosphatase conjugated goat anti-mouse antibody werepurchased from Bio-Rad Laboratories (Richmond, CA). [y- 3211ATP (3000Ci/mmol), myo- [2- 3H]inositol (18.3 Ci/mmol), and [ 3H]PAF (80 Ci/mmol) werepurchased from Amersham (Arlington Heights, IL). Scintillation fluid, 3-(N-morpholino)propanesulfonic acid (MOPS), and antibody PY-20 were purchased fromICN Biomedicals, Inc (Costa Mesa, CA). GF/C filters and P81 phosphocellulosepaper were purchased from Whatman (Mandel Scientific, Toronto, Canada). Fetalbovine serum (FBS), culture medium (F12 and DMEM), and antibiotics werepurchased from GIBCO (Grand Island, NY). DHC (137) and DIPC (140) weresynthesized as reported and stored in a stock solution of DMSO before serial dilutionin an appropriate buffer for each experiment. DMSO concentrations were below 0.2%in all experiments.302.2. PURIFICATION OF HUMAN NEUTROPHILS AND LYMPHOCYTES ANDMEMBRANE PREPARATION.Human blood from normal volunteers was obtained in heparin. Blood wasdiluted with Tyrode's buffer (137 mM NaC1 ,0.25 % gelatin, 2.68 mM KC1, 8 mMNa2HPO4 , 1.5 mM KH2PO4 , 11.9 mM NaHCO3, and 5.6 mM D-glucose, pH 6.5)(5:1, v/v) and red cells were sedimented for 30 min after mixing blood with dextransolution (6% dextran, 0.9 % NaC1) as reported (146). Neutrophils were isolatedfollowing Ficoll-hypaque gradient centrifugation and the remaining erythrocytes wereremoved by Tris/NH4C1 hemolysis (147). Neutrophils at greater than 97% purity andgreater than 95 % viability as assessed by Coulter Counter (Vancouver GeneralHospital, Hematology Dept.) and trypan blue dye exclusion, respectively.Lymphocytes were either purified from heparinized peripheral blood fromhuman volunteers or obtained as a pure fraction from the cell separator unit(Vancouver General Hospital). Peripheral blood was subjected to dextransedimentation and Ficoll-hypaque centrifugation as described above and thelymphocyte and platelet rich interface was collected and centrifuged at low speed(1,200 RPM in Beckman GPR centrifuge) to remove platelets. Cells were washedwith Tyrode's buffer (pH 6.5) and the platelet rich supernatant was discarded. Purifiedlymphocytes and neutrophils were collected in an appropriate buffer as specified forfurther analysis.For PAF receptor binding studies, membrane preparations or whole cells wereused to assess receptor affinity and receptor numbers, respectively. For membranepreparations, neutrophils were frozen in PAF binding buffer containing proteaseinhibitors (10 mM Tris-HC1 (pH 7.5), 0.25 % BSA, 5 mM MgC12, 10 mM KC1, 0.1mM PMSF, 1.46 j.IM pepstatin A, 42 p.M leupeptin, and 10 lig/m1 soybean trypsininhibitor). Cells were sonicated for 30 s and passed five times through a 26 gauge31syringe. The suspension was centrifuged for 10 min (2,000xg) at 4°C to removenuclei and intact cells. The broken cell supernatant fraction was layered onto 41 %sucrose and centrifuged at 30,000 RPM for 35 mM at 4°C in a TL-100 ultracentrifuge(Beckman). The membrane band was collected and centrifuged at 55,000 RPM for 20min at 4°C. The membrane pellet was suspended in binding buffer without BSA andthe protein content was determined by the method of Lowry (148).2.3. MEASUREMENT OF NEUTROPHIL ACTIVATION2.3.1. Immunoblotting with antiphosphotyrosine antibodiesNeutrophils (107 cells/m1) in Tyrode's buffer (pH 7.2) containing 1.4 mMCaC12 were treated at 37°C for 1 min with each agonist or pretreated with DHC priorto stimulation for 5 min at 37°C. After stimulation, cells were rapidly pelleted bymicrocentrifugation (14,000 RPM for 5 s), pellets were solubilized with 3 % Triton-X100 or sonicated in buffer composed of 50 mM Tris-HC1 (pH 7.7), 5 mM 13-methylaspartate, 150 mM NaC1, 0.2 mM Na3VO4 , 10 mM NaF, 1 mM NaMoO4 , 5mM EDTA, 10 µg/m1 leupeptin, 10 µg/ml soybean trypsin inhibitor, 1	 pepstatin,0.23 mM PMSF, and 1 mM diisopropylfluorophosphate. Insoluble cellular debriswere removed by microcentrifugation (14,000 RPM for 1 min) and soluble cellsupernatants were combined with SDS-PAGE sample buffer, boiled, andelectrophoresed by discontinuous SDS-PAGE. Separated proteins were transferred tonitrocellulose sheets and blocked overnight in tris-buffered saline (TBS) (0.02 MTris-HCI, pH 7.5, 0.05 M NaC1) containing 5 % BSA and 1 % ovalbumin. Blots werewashed with TBS and probed with antiphosphotyrosine specific antibody PY-20 inTBS with 1 % BSA and 0.02 % sodium azide. After washing, blots were incubatedwith alkaline phosphatase conjugated goat anti-mouse IgG in TBS + 0.05 % NP-40for 2 h at room temperature prior to color development with 5-bromo-4-chloro-3-32indolylphosphate (BCIP) and nitro blue tetrazolium (NBT). The specificity ofantibody PY-20 has been demonstrated by competition with phosphotyrosine orphenylphosphate but not phosphoserine, phosphothreonine, or free phosphate (149).2.3.2. Platelet-activating factor radioligand bindingCompetition binding studies were performed at room temperature asdescribed previously for platelet studies (150). Neutrophils and reagents weresuspended in binding buffer (10 mM Tris-HC1, pH 7.5, 10 mM KC1, 5 mM MgC12,and 2.5 mg/ml BSA) and 5 x 10 6 cells were added to start the reaction in tubescontaining 0.25 nM [3H]PAF and a 0 to 500-fold excess of unlabeled PAF for 30 minat room temperature. Reactions were terminated by filtration through prewetted GF/Cfilters, washed with 5 ml binding buffer and counted for radioactivity. Nonspecificbinding was referred to as the radioactivity associated with cells in the presence of a500-fold excess of unlabeled ligand.2.3.3. Polyphosphatidylinositide hydrolysisNeutrophils (2.5x107 cells/ml) were incubated with 100 p.Ci/ml of myo-[2-311]inositol in Tyrode's buffer (pH 7.2, no Ca 2+), for 90 min at 37°C. Cells werewashed twice and resuspended in Tyrode's buffer containing 12 mM LiC1, 1.3 mMCaCl2 and 0.6 mM MgC1 2. Cells in 0.5 ml volumes were incubated with or withoutDHC for 5 min and then treated with agonists for a further 5 mM. The reaction wasterminated by addition of 1.5 ml methanol/chloroform/HC1 (200:100:2) as reported(151). Following overnight storage at 4°C, water (0.6 ml) was added to each sampleto extract water-soluble inositol phosphates. From the upper phase, 1.8 ml wasdiluted with 2.5 ml of water and layered onto 1 ml Dowex anion exchange resincolumns (Bio-Rad) pre-equilibrated with water. Inositol phosphates were separatedand quantified as reported (152). Free inositol was eluted when the column was33washed with 16 ml 60 mM ammonium formate/5 mM disodium tetraborate; inositolmonphosphate (IP 1 ) was released with 6 ml of 200 mM ammonium formate/100 mMformic acid; inositol bisphosphate (1P 2) was eluted with 20 ml of 400 mM ammoniumformate/100 mM formic acid; and inositol trisphosphate (IP3) was recovered with 12ml of 1 M ammonium formate / 100 mM formic acid. 8 ml portions of column eluateswere counted for radioactivity and results were presented as the percentage of totalinositol phosphates recovered from the columns.2.3.4. Calcium MobilizationNeutrophils were suspended in phosphate buffer saline (PBS) (2.68 mM KC1,1.47 mM KH2PO4, 137 mM NaCl, 8.1 mM Na2HPO4, pH 7.4, no Ca 2+ or Mg2+)with 1 mM Fura-2/AM for 60 min at 37°C. Free Fura-2/AM was removed by washingthe cells three times with PBS (containing 0.9 mM CaC1 2 and 0.49 mM MgC12). Cells(5x106 cells/nil) were warmed to 37°C in a 1 cm 2 quartz cuvette in a volume of 2.5ml. Changes in Ca2+-dependent Fura-2 fluorescence were measured using a Perkin-Elmer LS-50 luminescence spectrophotometer with excitation wavelengths of 340and 380 nm and an emission wavelength of 510 nm (Norwalk, CT). The baselinelevel of fluorescence before addition of agonists was subtracted from agonist-inducedfluorescence in the presence and absence of drug. Values reported are arbitrary unitstaken from ratios between the absorbances at 340 nm and 380 nm.2.3.5. Protein kinase C assayNeutrophils (107 cells/m1) in Tyrode's buffer (pH 7.2) containing 1.4 mMCaC12 were stimulated with or without pretreatment with DHC and chemotacticfactors at 37°C. Treatments were terminated by quick centrifugation in a microfuge.Pellets were resuspended on ice in a sonication buffer (75 mMr3-glycerophosphate,20 mM MOPS (pH 7.2), 15 mM EGTA, 2 mM EDTA, 1 mM sodium orthovanadate34and 1 mM dithiothreitol) and sonicated for 30 s. The suspension was centrifuged at200,000 x g for 15 min in a Beckman TL-100 ultracentrifuge and the cytosolicsupernatant was aliquotted and frozen at -70°C. The pellet was resuspended in asonication buffer containing 1% NP-40, resonicated, subjected to TL-100ultracentrifugation, and the solubilized supernatant particulate fraction was stored at -70°C. Extracts were rapidly thawed and 0.5 mg of cytosolic and NP-40 solubilizedparticulate extracts were fractionated on a 1 ml anion exchange Mono Q columncoupled to a FPLC system (Pharmacia). Fractions (0.25 ml) were eluted at a flowrate of 0.8 ml/min using a linear gradient of 0 - 0.8 M NaC1 in a buffer containing 25mM p-glycerophosphate, 10 mM MOPS (pH 7.2), 5 mM EGTA, 2 mM EDTA, 1 mMsodium orthovanadate and 1 mM dithiothreitol. PKC activity of column fractionswere assayed by monitoring the transfer of 32P from [y32PJATP to substrates (153).Reactions were carried out for 10 min at 30°C in a volume of 25 41 with 50 pM ry-3211ATP (1500 cpm/pmol), 25 mM 0-glycerophosphate, 10 mM MOPS (pH 7.2), 15mM MgC12, 2 mM EGTA, 2 mM EDTA, 1 mM dithiothreitol, 500 nM PKIP, and 1mM sodium orthovanadate, with 1 mg/nil histone 111 or protamine chloride as thesubstrate. Reactions were initiated by the addition of [113211ATP and where stated,incubations included 4.5 mM CaC12, 60 i.tg/mlphosphatidylserine and 6 l_tg/m1 DAG.Reactions were terminated by spotting 20 p1 aliquots on 2 cm 2 pieces of WhatmanP81 phosphocellulose paper, dried 30 s, and washed with several changes of 1 %(v/v) phosphoric acid. Filter papers were transferred to vials, 3 ml Ecolume (ICN)scintillation fluid were added, and vials were counted for radioactivity in a Beckmanmodel LS5000 scintillation counter.2.3.6. Superoxide productionContinuous spectrophotometric measurement of a superoxide dismutaseinhibitable reduction of ferricytochrome C at 549 nm was measured in cuvettes as35described (154). Reaction mixtures containing 500 units/ml catalase and 1.24 mg/mlcytochrome C in Hank's balanced salt solution (HBSS) (1.3 mM CaC1 2, 5.4 mM KC1,0.44 mM MgC12, 0.41 mM MgSO4, 137 mM NaC1, 4.17 mM NaHCO 3 , and 0.34 mMNaHPO4, pH 7.4) supplemented with 5.5 mM glucose, 0.1 % gelatin and 1.5 - 2.5 x106 cells/ml. Prior to stimulation, mixtures were warmed to 37°C, agonists diluted inHBSS containing glucose and gelatin, and absorbance at 549 nm was scanned every10 s using a Beckman DU-65 spectrophotometer and plotted manually.To assess the kinetics of DHC inhibition of neutrophil stimulation, thesuperoxide assay was manipulated. Various cytochrome C and DHC concentrationswere varied in order to analyze the effect of this drug. To illustrate the kinetics ofinhibition, data was plotted according to Michealis and Menten (155) and Dixon(156). 02 generation was calculated using an extinction coefficient of 21.1 x 10 -3mol/litre/cm as nanomoles of cytochrome C reduced per minute by 10 6 cells minusSOD control.2.3.7. Elastase release[3H]elastin substrate was prepared as previously described using [3H]NaBH4as the radioactive source (157). The [ 3H]elastin suspension [specific activity=3168cpm/mg] was sonicated, agitated, and 20 suspensions were evenly spread on 16mm wells (Costar plates). The plates were dried at 45°C overnight, washed with PBS,and stored at 4°C until use (usually 1-7 weeks). Cells (10 6) in 500 DMEM wereadded to each well, stimuli or inhibitors diluted in DMEM were added, and plateswere incubated at 37°C in an atmosphere of 5% CO2. To monitor elastase release bythe cells into the media, 100 gl of media were removed after 1 h incubation,microcentrifuged (14,000 RPM for 2 min) to remove contaminating cells, and thesoluble phase was counted for radioactivity. Tritium counts from control wells36incubated with DMEM in the absence of cells were subtracted from wells with cellsand µg elastin degraded by 106 cells in 1 h was calculated.2.4. ANALYSIS OF PROTEIN-TYROSINE KINASE ACTIVITY IN CANINE'TRACHEAL SMOOTH MUSCLE CELLS2.4.1. Canine sensitization and guinea pig tracheal contraction assayMongrel dogs were sensitized by intraperitoneal immunization with 10 mg ofragweed pollen mixed with 30 mg Al(OH) 3 within 24 h of birth. Booster injectionswere administered weekly for 8 weeks and biweekly thereafter. After 5-6 months,the animals were sacrificed and the trachea were removed as reported (158). MaleCam-Hartley guinea pigs (300-400 g) were anesthetized with sodium pentobarbitaland sacrificed. Trachea were removed, cleaned, cut into spirals of 1 cm length, andplaced in oxygenated Kreb's-Henseleit solution (118 mM NaC1, 4.7 mM KC1, 1.2mM MgSO4 , 25 mM NaHCO3 , 1.2 mM KH2PO4, 11 mM glucose, and 3.3 mMCaCl2 , pH 7.5) in a jacketted organ bath under resting tension. Isometric contractionswere measured using a Grass 0.03 F force transducer and polygraph. Drugs wereadded to the organ bath in Kreb's buffer 10 min prior to the addition of agonists. Atthe end of each experiment carbachol (10 -3 M) was added to obtain maximalcontraction.2.4.2. Canine tracheal smooth muscle cell cultureTracheal smooth muscle strips were disected under a binocular microscope.The canine trachealis muscle provided a convenient source of smooth muscleproducing sufficient cell numbers to be used in this study. Smooth muscle cells weredispersed by agitated incubation in 0.2 % collagenase and 0.05 % elastase at 37°C for1-1.5 h, using a procedure modified from as previous report (159). Primary cell37cultures were maintained in DMEM/F12 (50:50) containing 10 % FBS. Using anti-a-actin smooth muscle specific antibodies, immunocytochemistry was performed inthe lab of our collaborator (Dr. N.L. Stephens, Manitoba) and smooth muscle cellswere found to be > 90 % purity. Confluent cultures of cells in 60 mm petri disheswere washed with PBS (no Ca 2+ or Mg2+) and dissociated with non-enzymatic celldissociation buffer for 30 min at 37°C. Dispersed cells were pelleted, resuspended inHBSS, counted, and diluted to 10 7 cells/ml. Cells were treated at 37°C and pelletedfor 15 s in a microfuge before solubilization in a suitable buffer.2.4.3. Preparation of smooth muscle cells extractsSmooth muscle cells were treated in HBSS at 37°C and rapidlymicrocentrifuged at 4°C. The cell pellet was resuspended in a sonication buffercomposed of 20 mM Hepes (pH 7.5), 100 IIM vanadate, 1 mM PMSF, 0.34 Msucrose, 2 mM P-mercaptoethanol, 1014/m1 leupeptin, 1014/m1 soybean trypsininhibitor, and 111M pepstatin. Cells were sonicated for 30 s on ice and unbroken cellsand nuclei were removed by centrifugation (2000xg, 10 min) at 4°C. Cytosolic cellextracts were recovered after centrifugation at 200,000 x g in a Beckman TL-100ultracentrifuge. The particulate pellet was then extracted in the same buffercontaining 0.5 mM EGTA, 2 mM EDTA, and 1 % NP-40. This suspension wasresonicated and subjected to ultracentrifugation as described above, and remainingsupernatants were referred to as particulate extracts. Protein content was determinedusing the method of Bradford (160) using bovine serum albumin as the standard.2.4.4. Protein-tyrosine kinase assayPhosphorylation of a synthetic copolymer of glutamate:tyrosine (poly(Glu;Tyr) 4:1) by smooth muscle cell extracts was determined using a modification of apreviously described procedure (75). The reaction volume was 50 IA containing 2038mM Hepes (pH 7.5), 10 mM MgC1 2 , 10 mM MnC12, 100 p,M sodium orthovanadate,7 mg/ml p-nitrophenylphosphate (as a competitive substrate for phosphatases (161)),201.1g of cell extract protein or 15 µ1 column fraction, 0.25 mg poly(Glu; Tyr) 4:1,and 8 1.IM ATP (2000-4000 cpm/pmolfy-32MTP). The reaction was initiated by theaddition of labelled ATP and incubated at 30°C for 10 min. The reaction wasterminated by spotting 40 gl on 2x2.5 cm pieces of P81 filter paper, air dried for 2min, washed overnight in 1% (v/v) phosphoric acid, and counted for radioactivitySpecific protein-tyrosine kinase activity was calculated by subtracting the 32Pincorporated into reactions without the copolymer from the 32P present in reactionscontaining the copolymer.2.4.5. Chromatography of smooth muscle cells extractsChromatography of extracts was performed using MonoQ and Superose 6columns coupled to a FPLC. The MonoQ anion exchange column was equilibratedwith 20 mM Hepes (pH 7.5), 1 mM PMSF, 2 mM 0-mercaptoethanol, 10011Mvanadate, 0.5 mM EGTA, 2 mM EDTA, and 7 µg/ml of each leupeptin, pepstatin A,and soybean trypsin inhibitor. Cytosolic and particulate protein (0.75 mg) was loadedonto the MonoQ column and 0.25 ml fractions were eluted at a flow rate of 0.8ml/min using a linear gradient of 0-0.8 M NaCl. Superose 6 gel filtrationchromatography was performed using the above buffer containing 100 mM NaCl.Extracted protein (0.35 mg) was loaded and eluted at a flow rate of 0.5 ml/min into0.6 ml fractions. Column fractions (15 p,1) were assayed for phosphorylation ofpoly(Glu;Tyr) 4:1 as described above.39STATISTICAL ANALYSISData are expressed as mean ± standard deviation. Elastase data was analyzedby a two-way analysis of variance using the Systat programme version 5.1 (Evaston,IL). Cellular inositol phosphate measurements were compared by Student's t test withcorrection for multiple comparisons.40CHAPTER 3RESULTS3.1. EFFECT OF TYRPHOSTINS ON AGONIST-INDUCED NEUTROPHILACTIVATION.3.1.1. Inhibition of human neutrophil protein-tyrosine phosphorylation.The ability of PAF, LTB4 and FMLP to stimulate tyrosine phosphorylation inhuman neutrophils was investigated. As seen by visual inspection of Figure 4A,unstimulated cells contained tyrosine phosphorylated proteins, although agoniststimulation caused an increase in the tyrosine phosphorylation of proteins of 41, 56,66, and 104 kDa. Preincubation of cells for 5 min with 0.2 mM DHC inhibitedligand-stimulated tyrosine phosphorylation of the proteins that were tyrosinephosphorylated in response to the agonists used. Protein-tyrosine phosphorylationwas reduced by drug treatment to the base line level seen in control, unstimulatedneutrophils. A dose-dependent inhibition of PAF-induced tyrosine phosphorylationwas observed from 0-0.1 mM DHC (Fig. 4B). Complete inhibition of PAF-inducedprotein-tyrosine phosphorylation was observed at 1014/m1 (42 [IM) in comparisonwith unstimulated neutrophils.In a separate study, crystal-induced neutrophil activation was analyzed.Degranulation and superoxide production from neutrophils activated by calciumpyrophosphate dihydrate (CPPD) crystals was inhibited by tyrphostins (data notshown; P.J.D., J. Jackson, H. Burt, and H. Salari, unpublished observations). Onantiphosphotyrosine immunoblots, increases in tyrosine phosphorylated substrateswere demonstrated in crystal-treated neutrophils that was enhanced by the presenceof serum. A different set of neutrophil proteins are tyrosine phosphorylated inresponse to crystal-induced activation compared with PAF, FMLP, and LTB 4 .41Crystal treatment increased tyrosine phosphorylation of substrates of 60, 51, 48 and43 kDa that was diminished by tyrphostin pretreatment (data not shown). Theseresults using a different protein-tyrosine kinase inhibitor clarify DHC results andsuggest that the effect of DHC is in fact the result of inhibition of protein-tyrosinekinase activity. Therefore, DHC serves as an appropriate probe to analyze protein-tyrosine kinase activity in neutrophils.42B 1 2 3 4 5 64341 4A10466456 441 ••110Oc.) 4C0.Ux■•• 66- 4343Figure 4 - Inhibition of tyrosine phosphorylation in neutrophils stimulated with PAF,LTB4, and FMLP by DHC. A. Human neutrophils (107 cells/ml) were preincubatedwith 0.2 mM DHC for 5 min prior to the addition of agonists (1 p,M) at 37°C for 1min. B. Dose-response of inhibition of PAF-induced protein-tyrosine phosphorylationof p41 by DHC. Cells were untreated (lane 1), treated with 1 j.t.M PAF for 1 min (lane2), or pretreated with 4.16 gM, 20.8 pM, 41.6 IA,M, and 104 .t.M DHC (lanes 3-6)prior to 111M PAF treatment. Cell extracts were separated by SDS-PAGE, transferredto nitrocellulose, and probed with antiphosphotyrosine antibodies. Molecular weightsare compared with I3-galactosidase (116 kDa), phosphorylase b (97 kDa), BSA (66kDa) and ovalbumin (43 kDa) are shown to the right and molecular masses oftyrosine phosphorylated substrates are shown to the left of the figure.3.1.2. Effect of DHC on ligand-receptor interactions.Studies with calcium release and 02 - generation indicated that the action ofDHC is directed against receptor mediated responses in neutrophils, perhaps due toinhibition of agonist-receptor interactions. To investigate this possibility, we testedthe effect of DHC on one of the agonists that binds to neutrophil membranes toinitiate cellular responses. PAF was selected for this study due to our lab's previousexperience with PAF receptor binding (150). Specific PAF receptor binding wasunaffected by the presence of 0.2 mM DHC as seen in Figure 5. In a singleexperiment, Scatchard plot analysis of data showed that human neutrophils possess-6000 receptors/cell with a Kd of -2.3 nM when neutrophils were incubated with 0.2mM DHC for 5 min prior to PAF binding. In the absence of the drug, the Kdremained 2.5 ± 0.2 nM with a Bmax of about 6000 receptors/cell (see Table 1). Theaffinity constant and number of receptor sites per neutrophil reported in this study arecomparable to previously determined values for high affinity PAF binding sites onperipheral blood neutrophils (64). These results suggest that DHC is not inhibitingPAF-induced responses at the receptor. Previous studies have demonstrated thaterbstatin does not inhibit FMLP binding (144), although the protein-tyrosine kinaseinhibitor ST638 dose dependently inhibited FMLP binding to neutrophils (75).44700cmc 	 600iic70500LLa. -a.c400=a-0en3001000Figure 5 - Lack of effect of DHC on PAF binding to human neutrophils. SpecificPAF binding to 5 x 106 neutrophils for 30 min at room temperature in the absence (A)and presence (B) of 0.2 mM DHC. Specific binding was defined as the differencebetween total binding of 0.25 nM [ 3H]PAF and the binding of 0.25 nM [3H]PAF inthe presence of a 500-fold excess (125 nM) unlabeled PAF. Triplicate determinationsare represented ± standard deviation.A 	 BTreatments45Table 1. Effect of DHC on PAF binding affinity and receptor number in humanneutrophils.Treatmenta 	 Kd (nM) 	 Bmax (receptors/cell)Control 	 2.5 	 6000208 RM DHC 	 2.3	 6000a Neutrophils (5 x 10 6) were incubated at room temperature with or without DHC for5 min and 0.25 nM [31-1113AF with 0 - 500 nM unlabeled PAF were added for 30 minA control study was performed to investigate the effect of lyso-PAF on PAFbinding to neutrophil membranes. In experiments where unlabelled PAF competedwith [311)13AF binding to neutrophil membranes, lyso-PAF did not effect PAFbinding. Using membranes and whole lymphocytes, a PAF receptor was identifiedthat had a similar affinity and receptor number than neutrophils. Using Scatchardplotting, a Kd of 1.63 nM and Bmax of 6,000 receptors/cell were determined.Although a PAF receptor has been demonstrated on a lymphocytic cell line (162), thisis the first identification of a PAF receptor on human lymphocytes. This suggests thatPAF may function to modulate immune responses and may activate these cells in theairways of asthmatic patients in allergic asthma.463.1.3. Effect of DHC on polyphosphatidylinositide hydrolysis.Receptor-induced activation of PLC catalyzes the hydrolysis of PIP 2, leadingto an accumulation of inositol trisphospate (IP3), inositol bisphosphate (IP2) andinositol monophosphate (IP 1 ). As an index of PLC activity, PLC products includinginositol mono, di, and triphosphates were monitored. Addition of 1 11M PAF and 11.1M FMLP were effective stimuli, increasing the production of all inositol phosphatesabove control levels (Fig. 6; I, II, and III, lanes c and g). Although PAF was a morepotent stimulus, the effects of both agonists were inhibited by pretreatment of cellswith DHC (Fig. 6; I, II, and III lanes d and h). LTB 4 (1 11M) slightly stimulated PIP2hydrolysis with IP 1 the only metabolite appreciably detectable above control whereasthe levels of 1P2 and IP3 were similar to unstimulated controls (Fig. 6, I, II, III, lanee). PI hydrolysis in all cases was reduced to levels obtained with untreated cells bydrug treatment. Incorporation of [311]-inositol into these water soluble phosphateswas not significantly affected in unstimulated cells by treatment with DHC (Fig. 6; I,II, and III, lane b) as compared with untreated control cells.47Figure 6 - Effect of DHC on agonist-induced polyphosphatidylinositide hydrolysis.Human neutrophils prelabeled with myo-[2- 3H]inositol were stimulated for 5 minwith 1 .tM LTB4 , PAF, or FMLP and the effect of a 5 min preincubation with 0.2mM DHC was analyzed for incorporation into (I) IP i (II) IP2 and (III) IP3 . Prior toharvesting and chromatography of inositol phosphates, cells were treated as follows:(a) untreated (b) DHC (c) FMLP (d) DHC + FMLP (e) LTB 4 (1) DHC + LTB4 (g)PAF (h) DHC + PAF. The incorporation of radioactivity into the variouspolyphosphatidylinositide metabolites was expressed as a percentage of the totalradioactivity associated with free inositol, IP, IP2, and IP3 together. Results are meanof 6 samples ± SD. Significantly inhibited compared to agonist-stimulated at**P<0.05; *P<0.1.Treatments483.1.4. The effect of DHC on ligand-induced increases in cytosolic free Ca 2+concentration.Since a rapid increase in intracellular free Ca 2+ is required for neutrophilactivation, the effect of this tyrosine kinase inhibitor on Ca 2+ release was analyzed. Inall cases, a calcium ionophore was used as a positive control and addition of bufferalone served as a baseline. Increases in intracellular Ca 2+ levels were observed inFura-2 loaded cells stimulated with PAF, FMLP, or LTB 4 (Fig. 7A). Maximumrelease of calcium with the three agonists were in the order LTB4>FMLP>PAF at aconcentration of 1 p.M. Pretreatment of neutrophils for 5 min with DHC caused adose-dependent decrease in Ca2+ mobilization in response to PAF, LTB 4 and FMLPby agonist stimulation of 10 -5, 10-7 , and 10-6 M, respectively (Fig. 7B). DHC waseffective when added as little as 1 min prior to the stimulus (data not shown). DHCdid not cause neutrophil toxicity as neutrophils washed with buffer after incubationwith DHC for 5 min were fully responsive to stimulation with the three agonists (datanot shown).49Figure 7 - Effect of DHC on intracellular calcium release. (A) Effect of agonistconcentration on intracellular Ca 2+ release. Fura 2 labeled cells were stimulated withvarious concentrations of PAF (■), LTB4 (0), and FMLP (A) and the change influorescence is plotted. Changes in fluorescence are reported as the difference influorescence before stimulation and at maximal fluorescence after stimulation. (B)Concentration dependent dose response curve of inhibition of intracellular Cali -release in human neutrophils by DHC in response to 101.1.M PAF (■), 0.111M LTB 4(0), and 1 .tM FMLP (A). Fluorescence is reported as the ratio between thatabsorbances at 340 nm and 380 nm. Results are reported as curves drawn by visualinspection and a representative of 3 similar experiments is illustrated.A60 -50 a) 50O Ac ......8Q 	 40 -4„) ...—a) >,o .7) 	30 -2 c.0- cpc 	 20 -N •L42= 	 1 0 'LL0 1 	 T 	 1 	 1 	 1 	 I 	 I-11 	 -10 	 -9 	 -8 	 -7 	 -6 	 -5 	 -4log [Agonist]B120a) -43-O toc c 100O 0O g2.COO 808 2O .;..• x 60astv E-■ 	 40f3 7„Lo..,c. 20..-.20050 	 100 	 150[DHC](I.tM)3.1.5. Inhibition of protein kinase C activation.PKC has been shown to play a major role in neutrophil activation asdemonstrated by the inhibition of neutrophil oxidative burst and degranulation byPKC inhibitors (100). In an attempt to characterize the site of action of DHC inneutrophils, the effect of this drug on PKC activity was investigated. The effect of 5min pretreatment of cells with DHC on agonist-induced PKC activity was analyzed.A rapid extraction of kinase activity was required to prevent proteolytic degradationof PKC during enzyme preparation. Neutrophils were treated with PAF (1 p.M) for 1min and extracts were isolated and separated by Mono Q FPLC chromatography. Thisfractionation was required, since PKC inhibitors have been found in cytosolic sourcesthat may be removed by anion exchange chromatography (163). Fractions wereassayed for PKC activity by monitoring Ca 2+ and phospholipid-dependentphosphorylation of histone H1 as well as the Ca 2+/DAG/PS-independentphosphorylation of protamine, a PKC substrate in the absence of activators (164).Most of the Ca2±/PS/DAG-dependent PKC activity (-60 %) was present in thecytosolic fraction and PKC was only slightly translocated to the NP-40 solubilizedparticulate fraction by PAF stimulation (Fig. 8B and 9B). The phosphorylation ofprotamine chloride was observed in the same fractions as those that phosphorylatedhistone Hl. Basal levels of PKC activity was associated with both cytosolic andparticulate fractions of control cells (Fig. 8A and 9A). Cytosolic extracts of untreatedneutrophils contained a single peak of PKC activity eluting at 0.33 M NaCl asseparated by MonoQ chromatography (Fig. 8A). PAF also stimulated the cytosolicPKC activity by approximately 1.5-fold (Fig. 8B). Pretreatment with DHC inhibitedthe cytosolic PKC activity to levels below baseline (Fig. 8C). Microsomal fractionscontained two peaks of PKC activity eluting at 0.33 M and 0.5 M NaC1 (Fig. 9A).Ca2+/PS/DAG dependent PKC activity in particulate derived fractions eluting at 0.33M was stimulated -1.5 fold by PAF (Fig 9B). After DHC pretreatment, PKC activity51stimulated by PAF treatment was reduced below control levels (Fig. 9C). DHCabolished the activity associated with the particulate 0.5 M NaC1 peak in theparticulate fraction, which was the major peak of PAF-induced activation. The Ca 2+and phospholipid dependence of phosphorylating activity eluting at 0.33 M NaC1 inboth cytosolic and microsomal fractions is exemplified by its inability tophosphorylate histone H1 in the absence of Ca2+, DAG, and PS. The particulate peakeluting at 0.5 M NaC1 was not dependent on Ca2+ or PS as similar activity was seenin the absence and presence of these activators (Fig. 9B). Similar results wereobserved with cells stimulated with FMLP and LTB4 (data not shown).5215001200900600300aO ---.= E 1500ccT: 1O a 1200=O .. Cen E 	 9000.c a-0. a_w 	 600030001500120090060030000.0 	 0 . 2 	 0.4 	 0 . 6 	 0 8[NaCI] (M)Figure 8 - Effect of DHC on PAF-induced activation of cytosolic PKC activity inhuman neutrophils. Treated or control cells were sonicated and rapidly centrifuged toobtain cytosolic extracts that were separated by Mono Q FPLC chromatographybefore being assayed for PKC activity. Cells were untreated (A), exposed to 1 .tMPAF for 1 min (B), or preincubated with 0.2 mM DHC for 5 min before 1 pM PAFstimulation for 1 min (C). Column fractions were assayed for phosphorylatingactivity of histone 111 in the absence (0) and presence (lb) of Ca2+, PS, and dioleinor protamine without activators (A). Similar results were obtained in threeindependent experiments.53---Figure 9 - Effect of DHC on PAF-induced activation of particulate PKC activity inhuman neutrophils. Particulate NP-40 solubilized fractions were assayed for PKCactivity and plotted as a function of NaC1 concentration as eluted off a Mono Q anionexchange column. Cells were untreated (A), exposed to 1 gM PAF for 1 min (B) orpreincubated with 0.2 mM DHC for 5 min before 11.1.M PAF stimulation for 1 min(C). Column fractions were assayed for phosphorylating activity of histone H1 in theabsence (0) and presence (•) of Ca2+, PS, and diolein or protamine withoutactivators (A). Similar results were obtained in three independent experiments.54400300200100aO ^0E 4003. Z-o 0-.cE300EO.co. w200CO 100'or) 	 E4-,	 O.Oo_ 040030020010000 . 0 	 0 . 2 	 0.4 	 0.6 	 0 . 8[NaCl] (M)3.1.6. Superoxide release.It has been reported that FMLP (165) and PAF (166) are capable of increasingsuperoxide production in human neutrophils. The effect of DHC on the generation ofsuperoxide anion was evaluated in neutrophils treated with 1 4M FMLP or PAF.When neutrophils were treated with FMLP and cytochalasin B (1 [tM) a rapidgeneration of superoxide (0 2) was observed. The 02 generation reached a plateau atabout 5 min after agonist treatment. Similarity, PAF in the presence of cytochalasin Bcaused a rapid generation of 02 from neutrophils. FMLP was a more potent stimulusfor 02- production (Fig 10). FMLP and PAF alone caused 02 release, although thepresence of cytochalasin B potentiated 02 release as reported (162). When cells werepretreated with 0.2 mM DHC 5 min prior to addition of FMLP or PAF, a potentinhibition of 02- generation was observed. The drug was able to inhibit 02- releaseby PAF or FMLP by greater than 70 % in several independent experiments (Fig. 10).55FMLPPAFFMLP + DHCPAF + DHCControl60.20 -0.10 -0.05 -0.002 	 3 	 4Time (min)0Figure 10 - Effect of DHC on stimulation of superoxide generation from humanneutrophils. Spectrophotometric analysis of agonist-induced reduction offerricytochrome C was monitored at 549 nm. Cells were either stimulated with 11.i.MPAF or FMLP in the presence of 1 gM cytochalasin B or cells were exposed to 0.2mM DHC for 5 min prior to addition of agonists. Control samples were untreated oragonist treated in the presence of superoxide dismutase. Representative data from 3similar experiments is shown.563.1.7. Elastase release.Neutrophil degranulation leads to the production of lysozomal enzymes andoxygen radicals. One component of lysozomal enzyme contents is elastase, a serineprotease that has been implicated in various disease states (167). In the presentinvestigation, the effect of DHC on the release of elastase from neutrophils wasevaluated. As has been reported (168), cytochalasin B appreciably augmentsneutrophil degranulation. In the absence of cytochalasin B, no significant increases inelastase release were detected in cells stimulated with FMLP, PAF, or LTB 4 (data notshown). In the presence of 1 pM cytochalasin B, a concentration previously shown tostimulate human polymorphonuclear leukocyte functions (146), 111M FMLPsignificantly stimulated elastase release from neutrophils (Fig. 11). Althoughcytochalasin B potentiated FMLP-induced elastase release, the presence ofcytochalasin B did not significantly enhance PAF or LTB 4-induced elastase release.A time course of elastase release from neutrophils indicated that a 1 h treatment timepermitted sufficient elastase secretion with little neutrophil death. Pretreatment ofneutrophils with DHC for 5 min prior to the addition of FMLP inhibited elastaserelease. The effect of DHC was dose-dependent (Fig. 12) and total inhibition ofFMLP-induced elastase release to control levels was seen at a drug concentration of83 4M. The IC50 for inhibition of elastase release was calculated to be about 35 p.M.It was observed that higher concentrations of DHC reduced non-specific elastaserelease, presumably due to stabilization of cellular activation processes. The basalrelease of elastase in untreated cells was also reduced below unstimulated levels byDHC (Fig. 11). Cells pretreated with the drug were viable after 1 h incubation timesas determined by a trypan blue exclusion dye assay.57*FMLP 	 PAF LTB 4Control•cn75	 15 —0Figure 11 - Inhibition of elastase release from human neutrophils by DHC.Neutrophils were preincubated with buffer alone (solid columns) or 0.2 mM DHC(open columns) for 5 min at 37°C. Elastase release in the presence of 1 JIMcytochalasin B was stimulated by 11.LNI of each agonist used. Vertical bars span thestandard deviation. The values obtained indicate that FMLP significantly stimulateselastase release in the presence of cytochalasin B (*, P 5_ 0.001, n=3). DHCsignificantly inhibits the release of FMLP-stimulated elastase release (#, P 5_ 0.001, n-,-. 3).Treatments58co 	 20w001-iia)Vi0 ) 	 10a)tsCc/iastsscoaFigure 12 - Dose-response curve of inhibition of FMLP-stimulated elastase releaseby DHC. Increasing amounts of DHC were incubated for 5 min at 37°C with 10 5-106neutrophils prior to 1 1.t.M FMLP stimulation (ID) or untreated cells (A). Solubilized[3H]elastin released into the culture medium was counted for radioactivity after a 1 hincubation. Vertical bars span the standard deviation. A representative of 3 similarexperiments is illustrated.0 	 50 	 100 	 150 	 200 	 250	300	350[DHC] (JIM )593.1.8. Kinetics of inhibition of neutrophil activation by DHC.The superoxide assay was used to determine the type of inhibition ofneutrophil responses by DHC. As seen in Fig. 13A, the velocity of superoxideformation increased with increasing concentration of cytochrome C present in thereaction. Thus, the substrate became saturated at higher cytochrome C concentrationsas the reaction velocity plateaued. Increasing the concentration of DHC caused adecrease in both Vmax and Km as seen in Table H. A decrease in Vmax and Km withincreasing inhibitor is characteristic of a competitive inhibitor, although the site ofinhibition is not known. From the Dixon plot in Figure 13B, a Ki for inhibition wascalculated to be 8 gM. Also, the reversibility of inhibition suggests that this is acompetitive inhibitor. Since this compound is a tyrphostin, it is likely that it competeswith the substrate to decrease activity, unlike genistein that inhibits ATP binding tothe enzyme.60•Figure 13 - Kinetic analysis of inhibition of FMLP-induced superoxide formation byDHC. A - Effect of increasing substrate concentration on the velocity of superoxiderelease illustrated by a Michealis-Menten plot. Cells were incubated at 37°C in HBSScontaining DHC at 0 (•), 52 (•), 104 (A) and 2081.IM (•). B - Dixon plot of theinhibition of superoxide production by DHC in the presence of cytochrome C. Therates of superoxide release (in nmoles 0 21min/106 cells) were calculated from valuesproduced by 5 min FMLP stimulation. Tile cytochrome C concentrations used were25 (0), 50 (A), 75 (0) .tM.61I ClS•a)1Ec20 	 40	60 	 80 	 100	120[Cytochrome C] (A1)[DHC]	 (gM)Table 2. Kinetic values for inhibition of superoxide production by DHC. DHCconcentrations of 0, 52, 104, and 20811M were used to determine Km and Vmaxvalues using the method of Lineweaver and Burk (169).[DHC] 	 Km 	 Vmax(PM) 	 (mM) 	 (nmoles 02 '/min/106 cells)0 1.36 4152 0.27 3.8104 0.24 2.1208 0.11 0.3623.2. IDENTIFICATION OF PROTEIN-TYROSINE KINASE ACTIVITY INAIRWAY SMOOTH MUSCLE CELLS.3.2.1. Inhibition of airway smooth muscle contraction by tyrphostins.We demonstrate that agonist-induced tracheal smooth muscle contraction canbe dimininshed by protein-tyrosine kinase inhibitors. As seen in Fig. 14 A, B and C,the contraction of guinea pig tracheal smooth muscle by mediators includinghistamine, leukotriene D4, and the muscarinic receptor stimulant carbachol can beinhibited by a protein-tyrosine kinase inhibitor, 4-hydroxy-3,5-diisopropylcinnamamide (DIPC). The presence of DIPC caused a decrease in theamount of contraction and a decrease in the sensitivity of contraction in response toeach agonist. This is illustrated by a decrease in the percentage of total contractionand a shift to the right, respectively, with increasing DIPC concentrations. To assessits effect, DIPC was added to the tissue 5 min prior to the addition of the agonists.The summary in Fig. 14 D shows the effect of DIPC on agonist-induced contractionby LTD4 , histamine, carbachol, endothelin-3, PDG 2, and TXA2. Also, ovalbumin-sensitized guinea pigs were used to analyze the effect of this inhibitor on antigenchallenged smooth muscle contraction. Maximal contraction was measured as thecontraction in response to 10 -3 M carbachol at the end of the experiment after thetissue had been washed and equilibrated. The inhibition of smooth muscle contractionwas not due to cell cytotoxicity, as this effect was reversible when the tissue waswashed.630 111	 •-10 	 -8Log [histamine]100806040200LTD4 hist carb *OA Endo PGD2 TXA2Treatment60B50 -40 -30 -20 -1 0-•	 • grit• •Figure 14 - Inhibition of guinea pig airway smooth muscle contraction in response to(A) histamine, (B) LTD4 , and (C) carbachol in the absence (71) and presence (•) of251.1.g/m1 4-hydroxy-3,5-diisopropylcinnamamide (DIPC). A summary of results in(D) shows the effect of 25 Kg/m1DIPC on contraction in response to 10 -6 M LTD4 ,10-4 M histamine (hist), 10 -3 M carbachol (carb), 10-9 M ovalbumin (OA), 10-6 Mendothelin-3 (endo), 10-5 M PGD2 , and 10-7 M TXA2. Contraction in response toagonists in the absence (dark columns) and presence (hatched columns) of 25 ms/m1DIPC are illustrated. Results are expressed as a percentage of the carbachol-inducedcontraction. Data are expressed as mean ± SD. *Designates ovalbumin sensitizedguinea pig trachea were used to assess ovalbumin-induced contraction.64-5• 1 	 • 	 • 	 j 	•	 i 	•	 •-9 	 -8 	 -7 	 -6 	 -5 	 -4 	 -3 	 -2Log [carbachol] (log M)-11 	 -10 	 -9 	 -8 	 -7 	 -6Log [LTD 4 ] (log M)3.2.2. Detection of tyrosine phosphoproteins by immunoblotting.Using immunoblotting, tyrosine phosphorylated substrates in cultured smoothmuscle cells were identified. Figure 15 shows multiple phosphorylated substrates insmooth muscle cells subjected to immunoblotting with an antiphosphotyrosineantibody. Phosphorylation on tyrosine residues of proteins of molecular weightranging from 34-180 kDa was observed. Long term (30 min) treatment with LTD 4 ,histamine, and carbachol had little effect on immunoreactivity withantiphosphotyrosine antibodies with the most heavily tyrosine phosphorylatedsubstrates at 46, 55, 106, and 111 kDa. Shorter treatments (1 min) stimulated protein-tyrosine phosphorylation of 40- and 73-kDa proteins when airway smooth musclecells were treated with histamine and carbachol (Fig. 15). Treatments of 5 min didnot detect increases in tyrosine phosphorylation of the 73 kDa phosphoprotein.Therefore, tyrosine phosphorylated proteins are present in resting airway smoothmuscle cells, although treatment with contractile agonists stimulates tyrosinephosphorylation in these cells.65Figure 15 - Protein-tyrosine phosphorylation of smooth muscle cell proteins detectedby immunoblotting with an antiphosphotyrosine antibody. Cells were untreated (1) ortreated for 1 min at 37°C with 0.1 mM histamine (2), and 0.1 mM carbachol (3) or 5min with histamine (4) and carbachol (5). Molecular weights of 13-galactosidase (116kDa), phosphorylase b (97 kDa), BSA (66 kDa) and ovalbumin (43 kDa) are shownto the left side of the figure and some of the molecular weights (in kDa) of tyrosinephosphoproteins are identified on the right.1	2	3	4	540,0 	Aumpagareimmumi,	 aftplenv 6673434406615000..-..iA 10000 -H ==. - 2.1..(.5 CtS......,3.2.3. Phosphorylation of poly(Glu,Tyr)4:1 by smooth muscle cell extracts.Figure 15 demonstrated the involvement of protein-tyrosine kinase activity insmooth muscle cells, therefore attempts were made too evaluate and characterize thisactivity in smooth muscle cells. Airway smooth muscle cell extracts were incubatedfor various lengths of time and the time dependence of poly(Glu,Tyr) 4:1phosphorylating activity is seen in Figure 16. Ten min incubation at 30°C wassufficient to demonstrate protein-tyrosine kinase activity in crude cytosolic and NP-40 particulate extracts from airway smooth muscle cells.Figure 16 - Time course of protein-tyrosine kinase activity in airway smooth musclecell extracts. Cytosolic (open boxes) and NP-40 solublized particulate (solid boxes)extracts (20 pg) were incubated f ,various times with the reaction buffer containing0.25 mg poly(Glu,Tyr) 4:1 and [ zPiATP. Results are expressed as mean values of arepresentative experiment.0	10 	 20 	 30 	 4067Time (min)683.3. PARTIAL PURIFICATION AND ANALYSIS OF PROTEIN-TYROSINE KINASEACTIVITY FROM AIRWAY SMOOTH MUSCLE CELLS.3.3.1. Effect of detergent on extraction of protein-tyrosine kinase activity.Figure 17 demonstrates protein-tyrosine kinase activity in the cytosolic andparticulate fractions extracted with various detergents. A rapid extraction was used toprevent proteolysis and loss of enzyme activity. The most protein-tyrosine kinase activitywas obtained from the particulate fraction using NP-40, and this detergent was used toprepare subsequent extracts.Figure 17 - Extraction of particulate protein-tyrosine kinase activity with various detergents.1 % Nonidet P40 (NP-40), 0.2 % Triton X-100 (TX-100), 1 mM cholate, and 1 mMdeoxycholate (DOC) were used to prepare smooth muscle cell extracts andpe activity iscompared to the cytosolic activity. Extracts from an equivalent of 2.5 x 10 cells wereassayed for protein-tyrosine kinase activity. A representative experiment is illustrated.Cytosol	NP -40 	 TX-100 	 Cholate	 DOCDetergent3.3.2. Effect of divalent cations on protein-tyrosine kinase activity.The effect of Mg2+ and Mn2+ concentration on smooth muscle protein-tyrosine kinase activities are shown in Figure 18. The activities of cytosolic extractsincreased with increasing divalent cation concentration up to 10 mM at which theactivity reached a plateau. Particulate NP-40 solubilized protein-tyrosine kinaseactivities increased to 20 mM with both cations, where an optimum activity wasobserved. The requirement for Mg 2+ and Mn2+ for activity is characteristic forprotein-tyrosine kinases although the optimal concentration has been shown to varydepending on the tissue source (170). Calcium chloride (4.5 mM) did not support theactivity in cytosolic or particulate fractions (data not shown).69Figure 18 - Effect of divalent cation concentration of phosphorylation of poly(Glu,Tyr)4:1 in cytosolic (A) and 1% NP-40 solubilized particulate (B) extracts. Specificprotein-tyrosine kinase activities were measured in the presence of the indicatedconcentrations of MgC12 (open boxes) and MnC12 (closed boxes). A representative oftwo similar experiments is illustrated.cncaC—•—cn	 8o E64200 	 10 	 20	30[Divalent cation] (mM)7014E 120.▪ 10 -F• 86 -0.O 4-E2-03.3.3. Thermal stability of protein-tyrosine kinases.To purify protein-tyrosine kinase activity from airway smooth muscle cells,the stability of this enzyme to heat was investigated. Figure 19 demonstrates that thestability of protein-tyrosine kinase activity after heating for 5 min at varioustemperatures. As expected, heat treatment at 37°C did not significantly alter protein-tyrosine kinase activity. Pretreatment at 50°C significantly diminished activity and80°C pretreatment completely abolished phosphorylation of poly(Glu, Tyr) 4:1 bysmooth muscle cell extracts. Frozen extracts and column fractions retained activitywhen thawed.Figure 19 - Stability of protein-tyrosine kinase activity after thermal treatment ofcytosolic (solid columns) and particulate (open columns) extracts prior to protein-tyrosine kinase assay. Extracts were rapidly thawed and kept on ice (0°C) or heatedfor 5 min at various temperatures as indicated. After heat treatment, extracts werekept on ice until assayed for phosphorylation of poly(Glu, Tyr) 4:1 as described in"Materials and Methods". Results of a single experiment are illustrated with verticlebars spanning S.D.0 	 37 	 50 	 80Temperature (°C)713.3.4. Anion exchange chromatography of cultured airway smooth muscle cellextracts.MonoQ anion exchange chromatography was first used to fractionate protein-tyrosine kinase activity from airway smooth muscle cells. Figure 20 demonstrates theisolation of protein-tyrosine kinase activity from cytosolic and particulate extractsusing MonoQ chromatography. In both extracts, one major peak of activity eluted at-0.32 M NaCl. In addition, minor peaks of tyrosine kinase activity eluted at -0.44 MNaC1 in the cytosolic fraction and -0.42 M and -0.55 M NaC1 in the particulatefraction.3.3.5. Gel filtration chromatography of cultured airway smooth muscle cellextracts.To estimate the molecular weight of protein-tyrosine kinase activity fromairway smooth muscle cells, Superose 6 fast protein liquid chromatography was used.Figure 21 illustrates the fractionation of cytosolic and NP-40 solubilized particulateextracts. Cytosolic extracts contained one major peak of poly(Gly;Tyr)4:1phosphotransferase activity with an estimated molecular weight of -60 kDa. A broadhigh molecular weight peak of protein-tyrosine kinase activity of -400-500 kDa witha shoulder peak at about 60 kDa eluted when particulate extracts were applied to theSuperose 6 column.72L 60,a.wN E5 50 -cn0 CLl■ %me,>., 	 40 -F...Figure 20 - MonoQ fast protein liquid chromatography of protein-tyrosine kinaseactivity extracted from cultured airway smooth muscle cells. After sonication andcentrifugation, 0.75 mg of cytosolic protein (A) and NP-40 solubilized particulateprotein (B) were chromatographed and fractions were assayed for poly(Glu, Tyr)4:1phosphotransferase activity. Similar results were obtained in three separateexperiments.7350A40 -30 -20 -ELia. 10-30 -20 -10-i. ■ • la mr-411 111111, 	 .	 4'al----m"--411B0 • • • 	 I	 I0.0 	 0.2 	 0.4	 0.6	0.8[NaCI] (M)4-3-wcnCuc..;4->a.•_E2-1.5 -1.0 -0.5 -•	0.0, 	 • 	 I	•	 I 	 •	 I 	 . 	 1	20 	 30 	 40 	 50 	 60Fraction70void 	 670 158 66 	 44■If 4, 41 	 ii87-AFigure 21 - Superose 6 fast protein liquid chromatography of protein-tyrosine kinaseactivity extracted from cultured airway smooth muscle cells. Cytosolic (A) and NP-40solubilized particulate (B) protein (0.35 mg) from untreated cultured airway smoothmuscle cells were separately fractionated and assayed for phosphotransferase activitytowards 0.25 mg poly(Glu;Tyr)4:1. Similar results were obtained in two separateexperiments.74CHAPTER 4DISCUSSION4.1. INHIBITION OF NEUTROPHIL RESPONSES BY DHC.Asthma is a complex airway disease that may involve multiple cells thatrespond directly to environmental conditions or indirectly via the production ofbiological response modifiers. Numerous reports suggest that different cell types areinvolved in airway hyperreactivity and asthma. Also, the involvement of numerousbiologically active molecules including inflammatory mediators, cytokines, andsecreted products have been implicated in the pathogenesis of asthma. Although theprecise mechanism of asthma is unclear, asthma has been well characterized as aninflammatory disease involving airway hyperresponsiveness. Therefore, theapproach of this study investigated the activation of neutrophils and smooth musclecells that are the primary cells involved with inflammation and airwayhyperresponsiveness, respectively.Protein-tyrosine kinases and their substrates have been associated primarilywith growth factors and are believed to play important roles in the onset ofoncogenesis (171). Our lab previously demonstrated protein-tyrosinephosphorylation in platelets in response to PAF and showed that a protein-tyrosinekinase inhibitor (erbstatin) can inhibit platelet activation (142). The effect of thisinhibitor was also investigated in neutrophils and found to decrease neutrophilbiological functions (144). Another protein-tyrosine kinase inhibitor, genistein, wasfound to inhibit PAF induced rabbit platelet aggregation and PLC activation at dosesgreater than 0.5 mM (143). Genistein was also found to inhibit human plateletresponses in response to stimulation with thromboxane A2 and collagen, but notthrombin (172). In leukocytes, various forms of protein tyrosine kinases such as c -fgr7 5(173), c-feslfps (174), c-hck (175) and c-src (176) have been identified. Themechanism of stimulation of these kinases and their role in cell function is largelyunknown. Recent work suggests that c-fgr in human neutrophils may be translocatedfrom granules to the plasma membrane upon degranulation (173). In the src genefamily, evidence suggests that phosphorylation of carboxy-terminal tyrosine residuesmay regulate their kinase activities (177). Pulido et al. (178) reported that aphosphotyrosine phosphatase is mobilized from specific and/or tertiary granules tothe plasma membrane fractions when neutrophils are stimulated. In addition, protein-tyrosine kinase activity has been demonstrated in neutrophil cytosolic and particulatefractions (75, 179).Huang et al. (180) reported that FMLP stimulated tyrosine phosphorylation ofseveral proteins with apparent molecular masses of group A (54-58 kDa and 100-125kDa) and group B (36-41 kDa) in rabbit neutrophils. In the present study, we foundthat FMLP stimulated tyrosine phosphorylation of 4 proteins of molecular masses ofapproximately 41, 56, 66 and 104 kDa. Similar phosphorylated proteins have beenreported to be present in neutrophils activated with PAF by other investigators (112).Also, similar but not identical proteins were found to be phosphorylated in humanneutrophils stimulated with GM-CSF (108), although GM-CSF also stimulatestyrosine phosphorylation of proteins of 78 and 92 IcD that may be the GM-CSFreceptor (110). The major human neutrophil tyrosine phosphorylated proteins as aresult of PAF stimulation were pp41, pp54, pp66, pp104 and pp116 (112).By using inhibitors of protein-tyrosine kinases or phosphatases, investigatorshave suggested that tyrosine phosphorylation may be a central process controllingneutrophil activation through receptor mediated processes (181). Although erbstatinwas able to inhibit superoxide production in human neutrophils stimulated by FMLP(144), this inhibitor failed to inhibit elastase release and intracellular calcium release.Since the inhibitor used in this study is more potent than erbstatin, this may account76for its ability to inhibit Ca2+ release. In addition, for erbstatin to inhibit 02 -production, it required at least 1 h of exposure for effective inhibition. These datasuggest that the action of erbstatin on 02 - production may not be specific and it ispossible that erbstatin was interfering with some other components of the superoxidegenerating system as well as protein-tyrosine phosphorylation. Several analogs oferbstatin have been synthesized (130, 131, 137) in an attempt to produce selective,non-toxic and potent inhibitors of protein-tyrosine kinases. The class of protein-tyrosine kinase inhibitors having a cinnamamide structure was first tested for theirability to inhibit tyrosine kinase activity in response to growth factors. The mostpotent of this class of compounds (a-cyano-3,4-dihydroxythiocinnamamide, DHC)was reported to inhibit EGF-dependent cell proliferation (182) with a Ki of 0.85 1.11V1(137), about six times more potent than erbstatin (129). In the present study, theeffect of DHC on human neutrophil responses was investigated. This work stemsfrom the observation that this inhibitor was the most potent protein-tyrosine kinaseinhibitor as reported in a previous study (137).This study demonstrates that DHC is capable of inhibiting agonist-inducedneutrophil activation. Responses such as 02 - generation and elastase release as wellas components of intracellular signaling pathways including Ca2+ release, PLC,PKC, and protein-tyrosine kinase activities were investigated. DHC partiallyinhibited TPA and zymosan induced 02 - generation when used at 0.2 mM (data notshown). These observations suggest that PKC activation and protein-tyrosine kinaseactivity are linked and that neutrophil phagocytosis involves protein-tyrosine kinaseactivation. To elucidate the site of action of DHC, a series of biochemical assayswere used to investigate the effect on calcium mobilization,polyphosphatidylinositide hydrolysis and PKC activation. It is well documented thatincreases in intracellular free calcium is associated with agonist-induced neutrophilactivation (183, 184). The ability of DHC to inhibit agonist-induced intracellular77Ca2+ accumulation suggests that the drug is acting by inhibiting PLC, possibly thesubstrate for an activated protein-tyrosine kinase. The inhibition of Ca 2+ release wasinhibited by short (5 min) preincubation with the drug and inhibition was reversible.When the drug was washed from the neutrophils, agonists were capable of inducingCa2+ release from the neutrophils suggesting that the drug did not cause cell toxicity.The yellow color of DHC was also decreased when cells were washed. Since theagonist-induced rise in cytosolic free Ca 2+ was inhibited by DHC, other parametersof neutrophil activation were analyzed.We discovered that the activities of PKC and PLC were decreased by thisdrug, suggesting that DHC was likely acting at a signal transduction process above orat the level of PLC. Therefore, the inhibition of PKC is the consequence of inhibitionof DAG and IP3-induced Ca2+ generation from PIP2 hydrolysis. In this study, PKCwas activated in both the particulate and cytosolic fractions, although a significanttranslocation of PKC to the membrane fraction did not accompany PKC stimulation.Since cytochalasin B was not present during stimulation, this correlates with reportsthat cytochalasin B is necessary for induction of particulate protein kinase activity(185). PKC inhibition below baseline levels may be due to a direct inhibitory effecton PKC as well as inhibiting upstream signals. Diminished PKC activity afterMonoQ chromatography suggests that DHC irreversibly binds to PKC to inhibit itsactivity below baseline levels. A nonspecific inhibition of signal transductionprocesses by DHC may also account for inhibition of all responses tested. A previousstudy correlates tyrosine phosphorylation and G-protein activation with therespiratory burst, indicating that kinases other than PKC are involved in this agonist-stimulated response (115). Our results support the claim that tyrosinephosphorylation may play an important role in neutrophil activation. The ability ofDHC to decrease PI hydrolysis to baseline levels indicates that this inhibitor iscapable of decreasing neutrophil stimulated activity initiated via PLC. DHC78pretreatment of cells does not decrease PI hydrolysis below baseline levels,suggesting that metabolism of unstimulated cells are not effected by this inhibitor.Leukocyte PLC activity has also been shown in immunoglobulin stimulated B cellstreated with tyrphostins and this inhibitor did not inhibit membrane-bound PLCactivity in vitro (145). These results support the claim that tyrosine phosphorylationmay be linked to PLC activation by the B cell antigen receptor that does not have aprotein-tyrosine kinase domain. On the other hand, tyrosine phosphorylation has alsorecently been shown to be involved in receptor coupling to phospholipase D inhuman neutrophils (186).The identity of the 41 kDa tyrosine phosphorylated protein in neutrophils isunknown, although several possibilities exist. It has been suggested by Gomez-Cambronero and coworkers (112) that a 40 kDa protein that is tyrosinephosphorylated in neutrophils is the G oa subunit of the heterotrimeric pertussissensitive G-protein. This scenerio indicates that tyrosine phosphorylation activatesthis G-protein subunit, although no findings support this possibility. Tyrosinephosphorylation of PLCy by receptor protein-tyrosine kinases has been shown toactivate polyphosphoinositide turnover, although tyrosine phosphorylation of otherPLC isozymes has not been demonstrated. In neutrophils activated withinflammatory mediators, it is possible that tyrosine phosphorylation activates a PLCand tyrphostins are capable of inhibiting activation at this level of signal transduction.In other systems, tyrosine phosphoproteins with 41-43 kDa have been determined tobe of the MAP kinase family of protein kinases and pp42 as well as pp44 MAPkinases have been cloned (187). It is possible that neutrophils, like other cells thathave increased tyrosine phosphorylation when activated, may employ a MAP kinaseat a pivotal point in its signal transduction mechanism. Interestingly, tyrosinephosphorylation of MAP kinase is stimulated by fluoroaluminate, suggesting thatactivated G-proteins initiate upstream events resulting in MAP kinase activation79(188). Although the above possibilities are conceivable, further experiments arerequired to clarify the unknown mechanism of neutrophil activation.In dogs, inhaled ozone and allergen challenge causes transient airwayhyperresponsiveness that is associated with neutrophil influx as determined byexamining BAL fluid (189). The pathogenesis of asthma involves the release ofoxygen radicals and enzymes that are released from neutrophils, damaging theairways. Reactive oxygen species and neutrophil granule contents containingmyeloperoxidase, elastase, collagenase, cationic proteins, and lysozyme and arehighly toxic and known to damage lung tissues (190). Therefore, results shown inthis study demonstrating inhibition of elastase and superoxide release are relevant toairway inflammation. Moreover, inhibition of neutrophil activation and release ofthese damaging substances may prevent epithelial cell damage and airwayhyperresponsiveness.4.2. IDENTIFICATION OF PROTEIN-TYROSINE KINASE ACTIVITY INAIRWAY SMOOTH MUSCLE CELLS.Although myosin phosphorylation plays a role in regulating smooth musclecontraction, a complex and poorly defined relationship has been shown to exist (191).Purified MLCK from tracheal smooth muscle is phosphorylated by cAMP-dependentprotein kinase, PKC, and calmodulin-dependent protein kinase II (121). Also, anumber of different protein kinases phosphorylate heavy chains or light chains ofsmooth muscle myosin, although only MLCK phosphorylates myosin light chains inresponse to either receptor-mediated stimulation or depolarization (123).Accordingly, several protein phosphatases have been identified and characterizedfrom bovine tracheal smooth muscle although a MLCK specific phosphatase has notbeen identified (122). Thus, there are many phosphorylating enzymes present in80smooth muscle cells and a complex circuitry is likely involved to regulate smoothmuscle contraction.Experimental animal models of asthma have been developed by sensitizinganimal to allergens. Guinea pigs repeatedly exposed to ovalbumin either byintraperitoneal immunization or aerosol develop hypersensitive airways that contractnon-specifically when exposed to various agents. Similarily, dogs sensitized byintraperitoneal immunization with ragweed pollen exhibit hypersensitivity andallergic bronchoconstriction with elevated IgE-mediated responses (192). Trachealsmooth muscle obtained from sensitized dogs has been shown to be altered bycomparison with littermate control dogs. Biochemical differences include increasedmyofibrillar adenosine triphosphatase (ATPase) activity (193) and increased myosinphosphorylation (194) that accompany a higher maximal velocity of shortening. Inaddition, electrophysiological parameters including the resting potential wasincreased in the airway smooth muscle of sensitized animals (195).Calcium is an important component of the signal transduction leading tosmooth muscle contraction. IP 3-induced calcium release from intracellular storesleads to smooth muscle contraction (196). Downstream of Ca 2+ release, severalmessengers and regulatory proteins have been implicated in coupling membranereceptors to the contraction of airway smooth muscle and schematic models havebeen proposed (76). Ultimately, control of cross-bridge cycling rates determines theproduction of force generation by airway smooth muscle. A continuing effort hasbeen to define the messengers and protein components that couple the activation ofmembrane receptors with smooth muscle contraction.In airway smooth muscle, the demonstration of decreased contraction bytyrphostins, tyrosine phosphorylation of proteins, and protein-tyrosine kinase activitymay lead to a better understanding of the mechanism of smooth muscle contraction.These results are supported by a recent report demonstrating vanadate (tyrosine81phosphatase inhibitor)-induced contraction and tyrosine phosphorylation in smoothmuscle (127). Demonstration of PDGF receptors on smooth muscle suggests thatprotein-tyrosine kinase activation may lead to smooth muscle proliferation (126).The increase in the amount of airway smooth muscle in human subjects with asthma(4) has been shown to be related to an increase in both the number (hyperplasia) andsize (hypertrophy) of the airway smooth muscle cells (197). Hypertrophy and/orhyperplasia may be cause wall thickening and exaggerate decreases in airway sizeproduced by smooth muscle contraction (125). Therefore, demonstration of protein-tyrosine kinase activity in airway smooth muscle cells suggests that this class ofenzyme may contribute to airway narrowing by acting on a component of thecontractile apparatus and/or by acting to mediate cell proliferation.Current asthma therapy is available that act as general antiinflammatoryagents at several sites to diminish symptoms. An understanding of the signaltransducing components of cells that contribute to the pathogenesis of asthma willlead to the production of more effective treatments. Several kinase inhibitors havebeen isolated and synthesized that are able to preferentially inhibit certain classes ofkinases. For example, several PKC inhibitors including staurosporine and moreselective inhibitors such as calphostin C have been studied (198). Also, numerousprotein-tyrosine kinase inhibitors have been synthesized and analyzed (130, 131,137). Recently, protein-tyrosine kinase inhibitors have been produced that are potentinhibitors of the EGF receptor kinase and only weakly inhibit the insulin receptorkinase (131, 137). This promising finding provides evidence that protein-tyrosinekinase inhibitors may be able to act preferentially on a particular kinase. Purifiedprotein-tyrosine kinases may be analyzed to determine structure/functionrelationships to develop selective inhibitors of specific kinases involved in aparticular cell function. Purified kinases may also be used to characterize82downstream substrates that lead to cell activation. Thus, a well characterized systemmaybe used to develop "designer drugs".4.3. RELATIONSHIP BETWEEN NEUTROPHIL ACTIVATION AND SMOOTHMUSCLE CONTRACTION.Inflammatory mediators are released from neutrophils that are responsible forthe development of airway narrowing in asthma. Using specific synthetase inhibitorsthat block mediator production, the presence of mediators during airwayhyperresponsiveness in lavage fluid was studies. Indomethacin, a cyclooxygenaseinhibitor, prevented ozone-induced airway hyperresponsiveness in dogs (199).Inhibition of cyclooxygenase prevents prostaglandin and thromboxane generation andimplicates these classes of mediators in airway hyperresponsiveness. Inhaled LTB 4in dogs increases TXA2 levels in lavage fluid during airway hyperresponsiveness andthe thromboxane synthesis inhibitor OKY-046 prevented thromboxane increases andairway hyperresponsiveness to LTB 4 (200). Further confirmation that thromboxanesmay be involved in airway hyperresponsiveness was demonstrated when the stablethromboxane mimetic U46619 increased airway responsiveness (37). TXA 2 not onlyincreases airway responsiveness (62), but also is capable of contracting airwaysmooth muscle by acting on specific thromboxane receptors (53). Stimulatedneutrophils have been shown to release thromboxane (201), although platelets arealso a source of thromboxanes. Higgs and collaborators (202) demonstrated thatneutrophils are the source of thromboxanes at inflammatory sites. Therefore, aconceivable link between neutrophil and smooth muscle cells has been established.As more sensitive assays for mediators become available, further links betweenneutrophils and smooth muscle cell activation may be clarified. In addition to theability of DHC to inhibit neutrophil release of elastase and superoxide, its effect on83thromboxane release would be an interesting investigation. Also, the effect ofactivated neutrophils on smooth muscle contraction in an organ bath may helpestablish a direct association between these cell types and their roles in inflammationand smooth muscle contraction.84Chapter 5CONCLUSIONThe present study demonstrated that an increase in protein tyrosinephosphorylation in neutrophils in response to chemotactic factors has an importantrole in neutrophil activation and DHC can specifically inhibit neutrophil activationwith relevance to inflammatory reactions. In addition, these studies showed thatDHC can inhibit neutrophil activation by interrupting tyrosine phosphorylation ofproteins that may have a key role in neutrophil function. Blocking release ofsuperoxide production and elastase release suggests that tyrosine phosphorylationlinks receptor activation with physiological responses in neutrophils. This inhibitorwas found to be a reversible and a competitive inhibitor of signal transductionprocesses that acts upstream of phospholipase C. Further analysis of this drug onneutrophil chemotaxis and mediator release will provide additional information onthis class of drugs as antiinflammatory agents. Studies using several inhibitors of thisclass may determine structure/function relationships and more selective neutrophilinhibitors may be synthesized. Also, the kinases that are responsible for neutrophilactivation may be characterized.The identification of protein-tyrosine kinase activity in smooth muscle cellsand its inhibition by tyrphostins suggests that these compounds may be used asprobes to analyze smooth muscle cells activation. Also, these compounds may beused in similar investigations as has been done in neutrophils to determine their siteof action. Purification and characterization of protein-tyrosine kinase activity fromsmooth muscle cells provides a site to prevent smooth muscle contraction involved inasthma and cardiovascular diseases. 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