CALCIUM AND cAMP AS INTRACELLULAR MESSENGERS

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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%).

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.

Spectrofluorimetric measurement of Ca2+ fluxes

Fura-2/AM (Calbiochem Corp., La Jolla, CA, USA) was used as the fluorescent, Ca2+­ sensitive indicator for these experiments. Neutrophils (1 x 1 07/ml) were pre-loaded with fura-2 (2 IJM) for 30 min at 3JOC in phosphate-buffered saline (PBS, 0.15 M, pH 7.4),washed twice and resuspended in indicator-free HBSS containing 1.25 mM CaCI 2 ,referred to hereafter as Ca2+-replete HBSS. The fura-2-loaded cells (2 x 1 06/ml) were then pre incubated with 1-HP (0.3-6.25IJM) for 10 min at 3JOC after which they were transferred to disposable reaction cuvettes, which were maintained at 3JOC in a Hitachi 650 1 OS fluorescence spectrophotometer with excitation and emission wavelengths set at 340nm and 500nm respectively. After a stable base-line was obtained (1 min), the neutrophils were activated by addition of FMLP ( 11JM) and the subsequent increase in fura-2 fluorescence intensity monitored over a 5 min period. The final volume in each cuvette was 3 ml containing a total of 6 x 106 neutrophils. Cytoplasmic da concentrations were calculated as described previously (Grynkiewicz et aI., 1985). Due to non-specific quenching of fluorescence at concentrations of 12.51JM and higher 25~M was the highest concentration of the pigment which could be used with the fura-2 system.

Radiometric assessment of Ca2 +fluxes

45Ca2+ (Calcium-45 chloride, specific activity 18.53 mCi/mg, Du Pont NEN Research Products, Boston, MA, USA) was used as tracer to label the intracellular Ca2 + pool and to monitor Ca2 + fluxes in resting and activated neutrophils. In the various assays of Ca2+ fluxes described below, including those of net efflux and influx, the radiolabeled cation was always used at a fixed, final concentration of 2 ~Cilml, containing 50 nmol cold carrier CaCI2. The final assay volumes were always 5 ml containing a total of 1 x 107 neutrophils. The standardisation of the procedures used to load the cells with 45Ca2+,as well as a comparison with silicone oil-based methods for the separation of labeled neutrophils from unbound isotope, have been described elsewhere (Anderson et a/.,1997).In the first series of experiments, neutrophils (2 x 106 /ml) were resuspended and equilibrated for 15 min at 3JOC in HBSS (final volume 5 ml) containing 45Ca2+ (2 ~Ci/ml) as the sole source of Ca2+ with and without 1-HP (12.5~M). The amount of cellassociated 45Ca2+ was then measured immediately prior to, and at 10, 20, 30, 60 and 90 sec, as well as 2, 3 and 5 min after the addition of FMLP (1IJM). Reactions were stopped by the addition of 10 ml Ca2 +-replete HBSS to the tubes which were transferred to an ice-bath (Anderson et a/., 1997). The cells were then pelleted by centrifugatiion at 400 g for 5 min followed by washing with 15 ml ice-cold Ca2 + -replete HBSS and the cell pellets finally dissolved in 0.5 ml of triton X-1 0010.1 M NaOH and the radioactivity assessed in a liquid scintillation spectrometer. Control, cell-free systems (HBSS and 45Ca2+ only) were included for each experiment and these values were substracted from relevant neutrophil-containing systems. These results are presented as the amount of cell-associated radiolabeled cation (pmoles 45Ca2+).

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
-IX3.3.3 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 ITS RELATIONSHIP 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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