The effects of 1-HP on elastase and MPO release

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CHAPTER 2 EXPOSURE OF FMLP-ACTIVATED HUMAN NEUTROPHILS TO THE Pseudomonas aeruginosa-DERIVED PIGMENT, 1-HYDROXYPHENAZINE (1-HP), IS ASSOCIATED WITH IMPAIRED CALCIUM EFFLUX AND POTENTIATION OF PRIMARY GRANULE ENZYME RELEASE

INTRODUCTION

Pyocyanine and 1-hydroxyphenazine (1-HP) are low molecular weight phenazine redox piigments produced by Pseudomonas aeruginosa (Ingram et a/. ,1970). Both pigments are present in the sputum of patients iinfected with this microbial pathogen and may contribute to both virulence and persistence by interfering with the mucociliary system (Wilson et a/., 1987; Wilson et a/., 1988; Munro et a/., 1989). Pyocyanine also inhibits epidermal cell growth (Cruickshank et a/., 1953) and lymphocyte proliferation (Nutman et a/., 1987), has antibiotic properties agaiinst other microorganisms (Schoental, 1941) and influences the acquisition of iron by P aeruginosa (Cox, 1986). 1- Hydroxyphenazine, but not pyocyanine, potentiates the release of the primary granule enzymes, myeloperoxidase (NlPO) and elastase, from activated neutrophils in vitro (Ras et a/., 1990; Ras et a/., 1992). This activity, if it is operative in vivo, would favour the development of chronic futile inflammatory responses, resulting in inflammationmediated tissue damage; this in turn would reduce host defenses and encourage microbial persistence, leading to a self-perpetuating cycle of bacteria-stimulated, hostmediated damage resulting in disease progression (Pier, 1985; Cole et a/., 1989). Although the pro-inflammatory interactions of 1-HP with human neutrophils have been described previously (Ras et a/. , 1990; Ras et a/. , 1992), the biochemical mechanisms by which these are achieved have not been elucidated. In the present study, the effects of 1-HP on the stimulus-activated increase in neutrophil cytosolic free Ca2+ levels,which precedes, and is also a prerequisite for extracellular release of primary granule enzymes (Knight et a/., 1982; Lew et a/., 1986; Nusse et a/., 1997), have been investigated in vitro. In addition, I have measured the levels of cyclic AMP, a second messenger which is intimately involved in the maintanance of Ca2+ homeostasis in excitable and non-excitable cells (Schatzmann, 1989; Johannsson et a/., 1992), in 1­ Hp-treated neutrophils.

METHODS

Preparation of the pigment, 1-Hydroxyphenazine (1-HP)

1-HP was prepared using procedures described in detail by Flood et a/. , 1972. Briefly,phenazine (500mg) was dissolved in 0.1 M HCI (1500ml) and photolyzed by plaCing the soiution 10cm below an overhead exposed fluorescent light for 3 days. 1-HP was extracted four times in 500ml of chloroform and then from the chloroform layer three times into 1 M NaOH (2500ml). The alkaline solution was acidified to pH 1.0 with acetic acid and 1-HP was re-extracted into chloroform. The chloroform layer was washed twice with 6% acetic acid and dried over anhydrous sodium sulfate, and the solvent removed under vacuum. 1-HP was obtained as a single substance as defined by highpressure liquid chromatography and characterized by ultraviolet spectrophotometry (maximum 273 in 0.1 M HCI), gas chromatography-electron impact mass spectrometry, and electrospray mass spectrometry (Watson et aI., 1986). 1-HP was stable with no loss of activity during incubation or prolonged refrigeration. For the experiments described below, 1-HP was dissolved in dimethyl sulfoxide (DMSO) to give a stock concentration of 10mM and uesd at a final concentration range of 0.3-12.5I1M with appropriate DMSO controls (maximum DMSO concentration of 0.125%).

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Chemicals and reagents

Unless indicated, all other chemicals and reagents were obtained from the Sigma Chemical Co, St Louis, MO, USA.

Preparation of neutrophils

Human neutrophils were obtained from heparinized (5U preservative-free heparin/ml) (Appendix 4) venous blood of healthy adult volunteers and separated from mononuclear leucocytes by centrifugation on histopaque-1 077 (Sigma Diagnostics, St. Louis, MO, USA) cushions at 400g for 25min at room temperature. The resultant cell pellet was suspended in phosphate-buffered saline (PBS) (0.151\11 (Appendix 3) at pH 7.4 and sedimented with 3% gelatin (Appendix 5) for 15min at 37°C to remove most of the erythrocytes. After centrifugation, residual erythrocytes were removed by selective lysis with 0.83% ammonium chloride (Appendix 1) at 4°C for 10min. The neutrophils, which were routinely of high purity (>90%) and viability (>95%) (Appendix 6), were resuspended to a concentration of 1 x1 07/ml in PBS and held on ice until ready for use.

Elastase and MPO release

Neutrophil degranulation was measured according to the extent of release of the primary granule-derived enzymes, elastase and myeloperoxidase (MPO). Neutrophils were incubated at a concentration of 1 x1 07/ml in indicator-free hank’s balanced salt solution (HBSS) with and without 1-HP (0.38-12.5IJM) for 1 Omin at 37°C. The stimulant, N-formyl-L-methionyl-L-Ieucyl-L-phenylalanine (FMLP, 1IJM), a synthetic chemotactic tripeptide, in combination with cytochalasin B (CB, 11JM) was then added to the cells,which were incubated for 15 min at 3JOC. The tubes were then transferred to an icebath, followed by centrifugation at 400g for 5min to pellet the cells. The neutrophil-free supernatants were then decanted and assayed for elastase and MPO activity using micro-modifications of conventional colorimetric procedures (Paul et al., 1978; Beatty et aI., 1982). In the case of elastase, 1251J1 of supernatant was added to the elastase substrate N-succinyl-L-alanyl-L-alanyl-L-alanine-p-nitroanilide, (3mM in 0.3% dimethyl sulphoxide DMSO) in 0.05M Tris-HCI (pH 8.0) and elastase activity monitored at a wavelength of 405 nm using a microplate spectrophotometer. In the case of MPO, neutrophil supernatants (201J1) were added to guaiacol and H20 2 (final concentrations of 10mM and 5mM respectively) in a final reaction volume of 250IJI and enzyme activity monitored spectrophotometrically at 450 nm.

SUMMARy 
SAMEVATTING 
ACKNOWLEDGEMENTS 
TABLE OF CONTENTS 
LIST OF FIGURES 
LIST OF TABLES 
LIST OF ABBREViATIONS
CHAPTER 1: LITERATURE REViEW 
1.1 INTRODUCTION
1.2 CYSTIC FIBROSiS 
2 PHAGOCYTES
3 PROTEASES
3.1 Neutrophil elastase (NE) 
3.2 Pseudomonas aeruginosa elastase (PE) 
4 PiGMENTS
4.1 Pyocyanine (Pyo)
4.2 1-hydroxyphenazine (1-HP)
4.3 Phenazine pigment production (Pyo and 1-HP)
4.4 Effects of pyocyanine and 1-HP on neutrophil function
4.5 Effects of pyocyanine, 1-HP and PE on ciliated respiratory epithelium
4.6 Effects of pyocyanine and 1-HP on lymphocyte function
4.7 Effects of pyocyanine on other cell types
5 CALCIUM AND cAMP AS INTRACELLULAR MESSENGERS
6 cAMP-BASED ANTI-INFLAMMATORY STRATEGIES
7 cAMP-ELEVATING AGENTS
7.1 Dibutyryl cAMP-vii7.1.1 Anti-inflammatory activities of dibutyryl cAMP
7.2 f32-adrenoreceptor agonists
7.2.1 Salmeterol and salbutamol..
7.2.1.1 Anti-inflammatory effects of salbutamol..
7.2.1.2 Anti-inflammatory effects of salmeterol..
7.3 Phosphodiesterase inhibitors
7.3.1 Theophylline
7.3.1.1 Anti-inflammatory effects of theophylline
7.3.2 Rolipram
7.3.2.1 Anti-inflammatory effects of rolipram
7.3.4 Adenosine receptor agonists
7.3.4.1 Adenosine A 1 receptors
7.3.4.2 Adenosine A2 receptors
7.3.4.3 Adenosine A3 receptors
7.3.4.4 Anti-inflammatory effects of A2a receptor agonists
8 THE AIM OF THE STUDY
CHAPTER 2: EXPOSURE OF FMLP-ACTIVATED HUMAN NEUTROPHILS TO THE Pseudomonas aeruginosa-DERIVED PIGMENT, 1-HYDROXYPHENAZINE (1-HP), IS ASSOCIATED WITH IMPAIRED CALCIUM EFFLUX AND POTENTIATION OF PRIMARY GRANULE ENZYME RELEASE 
2.1 INTRODUCTION
2.2 METHODS
2.2.1 Preparation of the pigment, 1-hydroxyphenazine
2.2.2 Chemicals and reagents
2.2.3 Preparation of neutrophils
2.2.4 Elastase and MPO release
2.2.5 Spectrofluorimetric measurement of Ca2+fluxes
2.2.6 Radiometric assessment of Ca2+ fluxes
2.2.6.1 45Ca2+-efflux out of FMLP-activated neutrophils
2.2.6.2 45Ca2+-influx into FMLP-activated neutrophils-VIII2.2.7 Radiometric assessment of Na+ influx
2.2.8 Intracellular cAMP levels
2.2.9 cAMP-dependent Protein Kinase A (PKA) activity
2.2.10 Intracellular ATP levels
2.2.11 Statistical analysis
2.3 RESUL TS
2.3.1 The effects of 1-HP on elastase and MPO release
2.3.2 The effects of 1-HP on the fura-2 responses of FMLP-activated neutrophils
2.3.3 45Ca2+ fluxes in activated neutrophils
2.3.4 Efflux of 45Ca2+ from FMLP-activated human neutrophils
2.3.5 Influx of 45Ca2+ into FMLP-activated human neutrophils
2.3.6 22Na+ fluxes in neutrophils
2.3.7 Intracellular cAMP levels
2.3.8 Intracellular A TP levels
2.3.9 cAMP-dependent protein kinase A (PKA)
2.4 DiSCUSSiON
CHAPTER 3: THE EFFECTS OF CONVENTIONAL INTRACELLULAR cAMPELEVATING AGENTS ON 1-HYDROXYPHENAZINE-MEDIATED INTERFERENCE WITH THE CLEARANCE OF CYTOSOLIC CALCIUM AND ENHANCEMENT OF ELASTASE RELEASE FROM FMLPACTIVATED NEUTROPHILS
3.1 INTRODUCTION
3.2 MATERIALS AND METHODS 
3.3 RESUL TS
3.3.1 Effects of the various cAMP-elevating agents on elastase release
3.3.2 Effects of the cAMP-elevating agents on 1-HP-mediated alterations in the fura-2 responses of FMLP-activated neutrophils
3.3.3 Effects of theophylline, salbutamol and salmeterol on neutrophil cAMP levels
3.4 DiSCUSSiON
CHAPTER 4: INVESTIGATION OF THE EFFECTS OF ADENOSINE RECEPTOR AGONISTS ON 1 HYDROXYPHENAZINE-MEDIATED ENHANCEMENT OF
RELEASE OF ELASTASE FROM ACTIVATED NEUTROPHILS AND ITSRELATIONSHIP TO ALTERATIONS IN INTRACELLULAR cAMP LEVELS
4.1 INTRODUCTION
4.2 MATERIALS AND METHODS 
4.3 RESUL TS
4.3.1 Effects of the adenosine receptor agonists on elastase release from FMLPICB-activated neutrophils
4.3.2 Effects of the A2a adenosine receptor antagonist (ZM241385) on CGS21680 and IB-MECA-mediated interference with 1-HP-induced enhancement of elastase release
4.3.3 Effects of CPA, CGS21680 and IB-MECA on neutrophil cAMP levels
4.4 DiSCUSSiON
CHAPTER 5: CONCLUSIONS 
5.1 Conclusions
REFERENCES
APPENDiCES
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