Exposure of N-Formyl-l-Methionyl-l-Leucyl-l-Phenylalanine-Activated Human Neutrophils to the Pseudomonas aeruginosa-Derived Pigment 1-Hydroxyphenazine Is Associated with Impaired Calcium Efflux and Potentiation of Primary Granule Enzyme Release

Abstract

The effects of pathologically relevant concentrations (0.38 to 12.5 μM) of the proinflammatory, Pseudomonas aeruginosa-derived pigment 1-hydroxyphenazine (1-hp) on Ca²⁺ metabolism and intracellular cyclic AMP (cAMP) in N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP; 1 μM)-activated human neutrophils, as well as on the release of myeloperoxidase (MPO) and elastase from these cells, have been investigated in vitro. Ca²⁺ fluxes were measured by the combination of a fura-2/AM-based spectrofluorimetric method and radiometric procedures, which together enable distinction between net efflux and influx of the cation, while radioimmunoassay and colorimetric methods were used to measure cAMP and granule enzymes, respectively. Coincubation of neutrophils with 1-hp did not affect intracellular cAMP levels or the FMLP-activated release of Ca²⁺ from intracellular stores but did retard the subsequent decline in the chemoattractant-induced increase in the concentration of cytosolic free Ca²⁺. These effects of 1-hp on the clearance of Ca²⁺ from the cytosol of activated neutrophils were associated with decreased efflux of the cation from the cells and increased release of MPO and elastase, while the delayed store-operated influx of the cation into the cells was unaffected by the pigment. The plasma membrane Ca²⁺-ATPase rather than a Na⁺-Ca²⁺ exchanger appeared to be the primary target of 1-hp. These observations suggest that the proinflammatory interactions of 1-hp with activated human neutrophils are a consequence of interference with the efflux of cytosolic Ca²⁺ from these cells.

Pyocyanin and 1-hydroxyphenazine (1-hp) are low-molecular-weight phenazine redox pigments produced by Pseudomonas aeruginosa (14). Both pigments are present in the sputum of patients infected with this microbial pathogen and may contribute to both virulence and persistence by interfering with the mucociliary system (20, 41, 42). Pyocyanin also inhibits epidermal cell growth (7) and lymphocyte proliferation (24), has antibiotic properties against other microorganisms (32), and influences the acquisition of iron by P. aeruginosa (6). 1-hp, but not pyocyanin, potentiates the release of the primary granule enzymes myeloperoxidase (MPO) and elastase from activated neutrophils in vitro (29, 30). This activity, if it is operative in vivo, would favor the development of chronic futile inflammatory responses, resulting in inflammation-mediated tissue damage; this in turn would reduce host defenses and encourage microbial persistence, leading to a self-perpetuating cycle of bacterially stimulated, host-mediated damage resulting in disease progression (5, 26).

Although the proinflammatory interactions of 1-hp with human neutrophils have been described previously (29, 30), 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 (16, 18, 23), have been investigated in vitro. In addition, we have measured the levels of cyclic AMP (cAMP), a second messenger which is intimately involved in the maintenance of Ca²⁺ homeostasis in excitable and nonexcitable cells (15, 31), in 1-hp-treated neutrophils.

MATERIALS AND METHODS

Preparation of 1-hp.

1-hp was prepared by procedures which have been described in detail elsewhere (9, 39, 40). Briefly, phenazine (500 mg) was dissolved in 0.1 M HCl (1,500 ml) and photolyzed by placing the solution 10 cm below an overhead exposed fluorescent light for 3 days. 1-hp was extracted four times in 500 ml of chloroform and then from the chloroform layer three times into 1 M NaOH (2,500 ml). The alkaline solution was acidified to pH 1.0 with acetic acid, and the 1-hp was reextracted into chloroform. The chloroform layer was washed twice with 6% acetic acid and dried over anhydrous sodium sulfate, and the solvent was removed under vacuum. 1-hp was obtained as a single substance as defined by high-pressure liquid chromatography and characterized by UV spectrophotometry (maximum, 273 nm in 0.1 M HCl), gas chromatography-electron impact mass spectrometry, and electrospray mass spectrometry (39). 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 10 mM and used at a final concentration range of 0.3 to 12.5 μM with appropriate DMSO controls (maximum DMSO concentration of 0.125%).

Chemicals and reagents.

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

Neutrophils.

Purified neutrophils were prepared from heparinized (5 U of preservative-free heparin/ml) venous blood of healthy adult human volunteers and separated from mononuclear leukocytes by centrifugation on Histopaque-1077 (Sigma Diagnostics) cushions at 400 × g for 25 min at room temperature. The resultant pellet was suspended in phosphate-buffered saline (PBS; 0.15 M; pH 7.4) and sedimented with 3% gelatin to remove most of the erythrocytes. After centrifugation, erythrocytes were removed by selective lysis with 0.84% ammonium chloride at 4°C for 10 min. The neutrophils, which were routinely of high purity (>90%) and viability (>95%), were resuspended to 10⁷/ml in PBS and held on ice until used.

Elastase and MPO release.

Neutrophil degranulation was measured according to the extent of release of the primary granule-derived enzymes elastase and MPO. Neutrophils were incubated at a concentration of 10⁷/ml in Hanks’ balanced salt solution (HBSS) with and without 1-hp (0.38 to 12.5 μM) for 10 min at 37°C. The stimulant, N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP; 1 μM), a synthetic chemotactic tripeptide, in combination with a submaximal concentration of cytochalasin B (CB; 1 μM) was then added to the cells, which were incubated for 15 min at 37°C. The tubes were then transferred to an ice bath, followed by centrifugation at 400 × g for 5 min to pellet the cells. The neutrophil-free supernatants were then decanted and assayed for elastase and MPO activity by micromodifications of standard colorimetric procedures (4, 25). Briefly, in the case of elastase, 125 μl of supernatant was added to the elastase substrate N-succinyl-l-alanyl-l-analyl-l-alanine-p-nitroanilide (3 mM in DMSO) in 0.05 M Tris-HCl (pH 8.0), and elastase activity was monitored at a wavelength of 405 nm. In the case of MPO, neutrophil supernatants (20 μl) were added to guaiacol and H₂O₂ (final concentrations of 10 and 5 mM, respectively) in a final reaction volume of 200 μl, and enzyme activity was monitored spectrophotometrically at 450 nm.

Spectrofluorimetric measurement of Ca²⁺ fluxes.

fura-2/AM (Calbiochem Corp., La Jolla, Calif.) was used as the fluorescent, Ca²⁺-sensitive indicator for these experiments. Neutrophils (10⁷/ml) were preloaded with fura-2 (2 μM) for 30 min at 37°C in PBS (0.15 M, pH 7.4), washed twice, and resuspended in indicator-free HBSS (pH 7.4) containing 1.25 mM CaCl₂, referred to hereafter as Ca²⁺-replete HBSS. The fura-2-loaded cells (2 × 10⁶/ml) were then preincubated with 1-hp (0.3 to 6.25 μM) for 10 min at 37°C, after which they were transferred to disposable reaction cuvettes, which were maintained at 37°C in a Hitachi 650 10S fluorescence spectrophotometer with excitation and emission wavelengths set at 340 and 500 nm, respectively. After a stable baseline was obtained (1 min), the neutrophils were activated by addition of FMLP (1 μM) and the subsequent increase in fura-2 fluorescence intensity was monitored for 5 min. The final volume in each cuvette was 3 ml containing a total of 6 × 10⁶ neutrophils. Cytoplasmic Ca²⁺ concentrations were calculated as described previously (12). Due to nonspecific quenching of fluorescence at concentrations of 12.5 μM and higher, 6.25 μM 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, Mass.) was used as a 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 μCi/ml, containing 50 nmol of cold carrier CaCl₂. The final assay volumes were always 5 ml containing a total of 10⁷ neutrophils. The standardization 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, has been described elsewhere (2).

In the first series of experiments, neutrophils (2 × 10⁶/ml) were resuspended and equilibrated for 15 min at 37°C in HBSS (final volume, 5 ml) containing ⁴⁵Ca²⁺ (2 μCi/ml) as the sole source of Ca²⁺ with and without 1-hp (12.5 μM). The amount of cell-associated ⁴⁵Ca²⁺ was then measured immediately prior to, and at 10, 20, 30, 60, and 90 s as well as 2, 3, and 5 min after, the addition of FMLP (1 μM). Reactions were stopped by the addition of 10 ml of Ca²⁺-replete HBSS to the tubes, which were transferred to an ice bath (2). The cells were then pelleted by centrifugation at 400 × g for 5 min followed by washing with 15 ml of ice-cold Ca²⁺-replete HBSS, the cell pellets were finally dissolved in 0.5 ml of Triton X-100–0.1 M NaOH, and the radioactivity was assessed in a liquid scintillation spectrometer. Control, cell-free systems (HBSS and ⁴⁵Ca²⁺ only) were included for each experiment, and these values were subtracted from those for the relevant neutrophil-containing systems. These results are presented as the amounts of cell-associated radiolabeled cation (pmoles of ⁴⁵Ca²⁺).

Efflux of ⁴⁵Ca²⁺ from FMLP-activated neutrophils.

To measure net efflux of ⁴⁵Ca²⁺ from neutrophils uncomplicated by concomitant influx of the radiolabeled cation, the cells (10⁷/ml) were loaded with ⁴⁵Ca²⁺ (2 μCi/ml) for 30 min at 37°C in HBSS. The neutrophils were then pelleted by centrifugation, washed once with and resuspended in ice-cold Ca²⁺-replete HBSS, and held on ice until use, which was always within 10 min of completion of loading with ⁴⁵Ca²⁺. By this procedure, the FMLP-activated fura-2 responses of neutrophils, similarly processed in HBSS containing 1 μM cold CaCl₂ followed by washing with and suspension in Ca²⁺-replete HBSS, did not differ from those of cells which had been maintained in Ca²⁺-replete HBSS throughout, indicating that at the time of measurement of efflux in the ⁴⁵Ca²⁺ system there was no meaningful depletion of intracellular Ca²⁺ (2). The ⁴⁵Ca²⁺-loaded neutrophils (2 × 10⁶/ml) were then preincubated for 10 min at 37°C in Ca²⁺-replete HBSS, in the presence and absence of 1-hp (1.55 to 12.5 μM), followed by activation with FMLP (1 μM) and measurement of the kinetics (10, 20, 30, and 60 s) of net efflux of ⁴⁵Ca²⁺. FMLP was omitted from the corresponding control systems. The reactions were terminated by the addition of 10 ml of ice-cold, Ca²⁺-replete HBSS to the tubes, and the cells were processed as described above.

Influx of ⁴⁵Ca²⁺ into FMLP-activated neutrophils.

To measure the net influx of ⁴⁵Ca²⁺ into FMLP-activated neutrophils, uncomplicated by concomitant efflux of the radiolabeled cation, the cells were loaded with cold, Ca²⁺-replete HBSS for 30 min at 37°C, after which they were pelleted by centrifugation, then washed once with and resuspended in ice-cold Ca²⁺-free HBSS, and held on ice until used. Preloading with cold Ca²⁺ was undertaken to minimize spontaneous uptake of ⁴⁵Ca²⁺ (unrelated to FMLP activation) in the influx assay. The efficiency of this loading procedure was demonstrated by measurement of the FMLP-activated fura-2 responses of the Ca²⁺-loaded neutrophils, which were similar to those of neutrophils maintained in Ca²⁺-replete HBSS (2). The Ca²⁺-loaded neutrophils (2 × 10⁶/ml) were then incubated for 10 min in the presence and absence of 1-hp (12.5 μM) at 37°C in Ca²⁺-free HBSS followed by simultaneous addition of FMLP and ⁴⁵Ca²⁺ (2 μCi/ml) or ⁴⁵Ca²⁺ only to control, unstimulated systems. The kinetics of influx of ⁴⁵Ca²⁺ into FMLP-activated neutrophils were then monitored over 5 min and compared with those of influx of the radiolabeled cation into the identically processed, unstimulated cells.

Radiometric assessment of Na⁺ influx.

Neutrophils (2 × 10⁶/ml) were suspended in 50 mM HEPES-Tris buffer supplemented with 135 mM choline chloride, 1.1 mM glucose, 1.8 mM CaCl₂, 0.8 mM MgSO₄, 5 mM KCl, 1 mM KH₂PO₄ and 100 μM cold NaCl, containing 0.5 μCi of ²²Na⁺ (sodium-22; specific activity, 398.99 mCi/mg; Du Pont NEN Research Products) per ml with and without 1-hp (12.5 μM) in a final volume of 5 ml (27) at 37°C. Thereafter, 100 μl of FMLP (1 μM final concentration) or an equal volume of buffer was added to each tube, and the amount of cell-associated ²²Na⁺ was measured over a time course ranging from 10 s to 5 min in control and stimulated cells. Appropriate background values (cells plus ²²Na⁺ with or without FMLP maintained at 4°C throughout the entire time course of the experiment) were included. Reactions were terminated by the addition of ice-cold PBS, processed as described above for ⁴⁵Ca²⁺ efflux-influx experiments, and the amount of cell-associated ²²Na⁺ was determined with an LKB Wallac 1261 Multigamma Counter (Turku, Finland) following lysis of the cells with 0.5 ml of Triton X-100–0.1 M NaOH.

Intracellular cAMP levels.

Neutrophils at a concentration of 10⁷/ml in HBSS were preincubated for 10 min at 37°C with and without 1-hp (12.5 μM). Following preincubation, the cells were treated with 1 μM FMLP (stimulated cells) or an equal volume of HBSS (resting cells) in a final volume of 1 ml, and the reactions were terminated and the cAMP was extracted by the addition of ice-cold ethanol (65% [vol/vol]) at 20 s and 1, 3, and 5 min after addition of the stimulant. The resultant precipitates were washed twice with ice-cold ethanol, and the supernatants were pooled and centrifuged at 2,000 × g for 15 min at 4°C. The supernatants were then transferred to fresh tubes and evaporated at 60°C under a stream of nitrogen. The dried extracts were reconstituted in assay buffer (0.05 M acetate buffer, pH 5.8) and assayed for cAMP by using the Biotrak cAMP ¹²⁵I-scintillation proximity assay system (Amersham International plc, Little Chalfont, Buckinghamshire, United Kingdom), which is a competitive binding radioimmunoassay procedure. These results are expressed as picomoles of cAMP per 10⁷ neutrophils. Because cAMP is rapidly hydrolyzed in neutrophils by phosphodiesterases, these experiments were performed both in the absence and in the presence of 1 μM rolipram (kindly supplied by M. Johnson, GlaxoWellcome plc, Stockley Park West, London, United Kingdom), a selective type 4 phosphodiesterase inhibitor, the predominant type found in human neutrophils (36).

Intracellular ATP levels.

The intracellular ATP levels were measured in the lysates of neutrophils (2 × 10⁶/ml) which had been incubated for 30 min at 37°C in the presence and absence of 1-hp (12.5 μM), by a sensitive luciferin-luciferase chemiluminescence procedure (13). These results are expressed as nanomoles of ATP per 10⁷ neutrophils.

cAMP-dependent protein kinase A (PKA).

The effects of 1-hp on the activity of PKA were measured by using the Pierce colorimetric PKA assay kit, Spinzyme format (Pierce, Rockford, Ill.). Briefly, PKA (0.5 U of purified catalytic subunit from bovine heart [Pierce]) was coincubated with 1-hp (12.5 μM) for 10 min at 30°C followed by addition of a synthetic peptide substrate labeled with a fluorescent probe, in a final volume of 25 μl of assay buffer containing 2 mM ATP and 100 mM cAMP. After incubation at 30°C for 30 min, phosphorylated and nonphosphorylated substrates were separated on an affinity membrane, which selectively binds the phosphorylated peptide. The membranes were washed, and bound peptide was eluted and assayed spectrophotometrically at 570 nm.

Statistical analysis.

The results of each series of experiments are expressed as the mean values ± standard errors of the means (SEM). Levels of statistical significance were calculated by Student’s t test when two groups were compared or by analysis of variance with subsequent Tukey-Kramer multiple comparisons test for multiple groups. The computer-based software systems Instat II and Minitab were used for analyses. Significance levels were taken at a P value of <0.05.

RESULTS

1-hp effects on elastase and MPO release.

The results of experiments determining 1-hp effects on elastase and MPO release are shown in Fig. 1. 1-hp caused dose-related enhancement of release of elastase and MPO by FMLP-CB-activated neutrophils which achieved statistical significance at concentrations of 1.5 and 3.1 μM and upwards for elastase and MPO, respectively. At concentrations of 3.1, 6.25, and 12.5 μM, the effects of 1-hp on release of elastase from FMLP-CB-activated neutrophils were significantly greater than those at concentrations of 0.38, 0.77, and 1.55 μM (P < 0.001) but were not different from each other. In the case of MPO release, concentrations of 6.25 and 12.5 μM 1-hp were significantly more potent (P < 0.001) than lower concentrations of the pigment (0.38 to 1.55 μM) but did not differ significantly from each other in effect. The magnitude of enhancement of MPO release observed at 12.5 μM 1-hp was only slightly greater than that observed at 6.25 μM, which is probably due to assay interference as a result of the HOCl-scavenging properties of the pigment at concentrations in excess of 6.25 μM (7). The pigment did not affect the release of either elastase or MPO from unstimulated cells (data not shown).

FIG. 1.

Effects of varying concentrations of 1-hp (0.38 to 12.5 μM) on the release of elastase and MPO from FMLP-CB-activated neutrophils. The results of a typical experiment with 12 replicates for control and pigment-treated systems are presented as the mean percentages ± SEM of the values for the corresponding pigment-free control systems. ∗, P < 0.05 to 0.001.

1-hp effects on the fura-2 responses of FMLP-activated neutrophils.

These results are shown in Fig. 2 and Table 1. The results shown in Fig. 2 are traces from three typical experiments, which depict the effects of 6.25 μM 1-hp on the fura-2 responses of FMLP-activated neutrophils. Addition of FMLP to neutrophils was accompanied by the characteristic, abrupt increase in fura-2 fluorescence due to an increase in the cytosolic concentration of Ca²⁺. While 1-hp did not affect the abrupt increase in fluorescence intensity, pretreatment of FMLP-activated neutrophils with the pigment retarded the speed of the subsequent decline in fluorescence, indicative of interference with clearance of Ca²⁺ from the cytosol.

FIG. 2.

FMLP-activated fura-2 fluorescence responses of control (solid lines) and 1-hp (6.25 μM)-treated (dashed lines) neutrophils. FMLP was added as indicated (arrows) when a stable baseline was obtained (±1 min). The traces shown are from three different experiments.

TABLE 1.

Peak cytosolic calcium concentrations ([Ca²⁺]_i) and time taken for these to decline to half-peak values in FMLP-activated control and 1-hp-treated neutrophils^a

System	Peak [Ca2+]i value (nM)	Time taken to decline to half-peak value (min)
FMLP only (control)	849 ± 97	1.10 ± 0.21
FMLP + 1.6 μM 1-hp	848 ± 82	1.14 ± 0.17
FMLP + 3.1 μM 1-hp	837 ± 48	1.40 ± 0.10b
FMLP + 6.25 μM 1-hp	785 ± 75	1.50 ± 0.12b

^aThe results of 10 experiments are expressed as the mean values ± SEM. The [Ca²⁺]_i value for unstimulated neutrophils was 127 ± 14 nM. 

^bP < 0.001 for comparison with the 1-hp-free control system. 

The results shown in Table 1 are those from a larger series of experiments and show peak cytosolic Ca²⁺ concentrations ([Ca²⁺]_i), as well as the time taken for fluorescence intensity to decline to half-peak (t_1/2) values, for neutrophils activated with FMLP in the presence and absence of varying concentrations of 1-hp. As indicated above, 1-hp, at the concentrations used, did not affect the abruptly occurring increase in cytosolic [Ca²⁺]_i following activation of neutrophils with FMLP. However, the pigment at concentrations of 3.1 μM and upwards significantly prolonged the time taken for fluorescence to decline to half-peak values.

⁴⁵Ca²⁺ fluxes in activated neutrophils.

The time course of ⁴⁵Ca²⁺ fluxes in control and 1-hp (12.5 μM)-treated, FMLP-activated neutrophils maintained at 37°C in HBSS containing ⁴⁵Ca²⁺ throughout the experiment is shown in Fig. 3. Following exposure of the control cells to FMLP, there was an abrupt decrease in the amount of neutrophil-associated ⁴⁵Ca²⁺ which terminated at about 30 s and resulted in a mean loss of 33% of the radiolabeled cation. This was followed by an initial slow recovery in the amount of cell-associated ⁴⁵Ca²⁺ (1 to 2 min) and by accelerated uptake of the cation thereafter (3 to 5 min) which was complete at 5 min. The amount of ⁴⁵Ca²⁺ released from 1-hp-treated neutrophils 30 s after the addition of FMLP was significantly less (P < 0.02) than that released from control neutrophils, while the subsequent rate and extent of uptake appeared similar. However, the apparent decrease in efflux of ⁴⁵Ca²⁺ in 1-hp-treated cells in the setting of unaltered uptake resulted in poststimulation intracellular concentrations of the cation which were higher than those of the pigment-free control cells (P < 0.02).

FIG. 3.

Fluxes of ⁴⁵Ca²⁺ following exposure of neutrophils to FMLP in the absence (solid circles) and presence (open circles) of 12.5 μM 1-hp. The results of seven different experiments are expressed as the mean amounts of cell-associated ⁴⁵Ca²⁺ (picomoles per 10⁷ cells) ± SEM. ∗, P < 0.02.

Efflux of ⁴⁵Ca²⁺ from FMLP-activated neutrophils.

In the experiments which were designed to measure net efflux of ⁴⁵Ca²⁺ from FMLP-activated neutrophils uncomplicated by concomitant influx, cells which had been preloaded with ⁴⁵Ca²⁺ and then washed and transferred to Ca²⁺-replete HBSS (to minimize reuptake of radiolabeled cation) were activated with FMLP in the presence and absence of 1-hp (1.6 to 12.5 μM) followed by measurement of the amount of cell-associated ⁴⁵Ca²⁺. The kinetics of net efflux of ⁴⁵Ca²⁺ from neutrophils activated with FMLP in the presence and absence of 12.5 μM 1-hp are shown in Fig. 4. Addition of FMLP to neutrophils resulted in an abrupt efflux of the cation, which terminated at about 30 s after addition of the chemoattractant. Treatment of neutrophils with the pigment resulted in a statistically significant decrease (P < 0.002 at 60 s) in the rate and extent of efflux of ⁴⁵Ca²⁺. The results of a series of experiments in which the effects of varying concentrations of 1-hp on the efflux of ⁴⁵Ca²⁺ from FMLP-activated neutrophils were investigated by using a fixed 60-s incubation period are shown in Table 2.

FIG. 4.

Kinetics of efflux of ⁴⁵Ca²⁺ from resting (solid triangles) and FMLP-activated neutrophils in the absence (solid circles) and presence (open circles) of 12.5 μM 1-hp. The results of nine different experiments are expressed as the mean amounts of cell-associated ⁴⁵Ca²⁺ (picomoles per 10⁷ cells) ± SEM. ∗, P < 0.002.

TABLE 2.

Effects of varying concentrations (3.1 to 12.5 μM) of 1-hp on the efflux of ⁴⁵Ca²⁺ from FMLP-activated neutrophils^a

System	Amt of 45Ca2+ released from neutrophils 60 s after addition of FMLP (pmol/107 cells)
FMLP only	155 ± 10
FMLP + 3.1 μM 1-hp	145 ± 5
FMLP + 6.25 μM 1-hp	120 ± 5b
FMLP + 12.5 μM 1-hp	95 ± 5b

^aThe results of six experiments are expressed as the mean values ± SEM. 

^bP < 0.02 to 0.0001 for comparison with the 1-hp-free control system. 

Influx of ⁴⁵Ca²⁺ into FMLP-activated neutrophils.

For the experiments determining influx of ⁴⁵Ca²⁺, neutrophils were preloaded with cold Ca²⁺ and then transferred to Ca²⁺-free HBSS prior to activation with FMLP, which was added simultaneously with ⁴⁵Ca²⁺. The results of these experiments, designed to measure net influx of ⁴⁵Ca²⁺ into FMLP-activated neutrophils in the presence and absence of 1-hp, are shown in Fig. 5. Activation of control, pigment-free neutrophils with FMLP under these experimental conditions resulted in a delayed uptake of ⁴⁵Ca²⁺, which occurred after a lag phase of 30 to 60 s. Influx of ⁴⁵Ca²⁺ appeared to be a true consequence of activation of neutrophils with FMLP since there was only trivial influx of the radiolabeled cation over the same time course into control, identically processed neutrophils which had not been exposed to FMLP. Pretreatment of neutrophils with 12.5 μM 1-hp did not detectably alter the extent of influx of ⁴⁵Ca²⁺ into FMLP-activated neutrophils.

FIG. 5.

Kinetics of influx of ⁴⁵Ca²⁺ into unstimulated (solid triangles) and FMLP-activated neutrophils in the absence (solid circles) and presence (open circles) of 1-hp (12.5 μM). The results of three different experiments are expressed as the mean uptakes of ⁴⁵Ca²⁺ (picomoles per 10⁷ cells) ± SEM.

²²Na⁺ and neutrophils.

Intracellular concentrations of ²²Na⁺ were extremely low in unstimulated neutrophils and were unaffected by the addition of FMLP throughout the 5-min time course of the experiment. At 30 s and 5 min after the addition of FMLP (times which corresponded with maximum efflux and influx of ⁴⁵Ca²⁺, respectively), the respective amounts of cell-associated ²²Na⁺ following correction for background values were 178 ± 12 and 144 ± 13 pmol of ²²Na⁺/10⁷ cells. The corresponding value for unstimulated cells immediately prior to the addition of FMLP was 169 ± 15 pmol of ²²Na⁺/10⁷ cells (data from three separate experiments).

Intracellular cAMP levels.

The results for intracellular cAMP levels are shown in Table 3. Exposure of resting neutrophils to 1-hp (12.5 μM) in the presence or absence of rolipram caused an approximate doubling in intracellular cAMP levels, while those in FMLP-activated cells, although higher than in resting cells, were unaffected by the pigment.

TABLE 3.

Intracellular cAMP Levels in unstimulated and FMLP-stimulated neutrophils in the presence and absence of 1-hp^a

System	Intracellular cAMP (pmol/107 cells)
Neutrophils only	14 ± 3
Neutrophils + 1 μM rolipram	64 ± 32
Neutrophils + 12.5 μM 1-hp	32 ± 15
Neutrophils + rolipram + 1-hp	126 ± 42
FMLP-activated neutrophils	49 ± 11
FMLP-activated neutrophils + 1-hp	56 ± 28
FMLP-activated neutrophils + rolipram	145 ± 50
FMLP-activated neutrophils + rolipram + 1-hp	155 ± 64

^aThe results of four experiments are expressed as the mean values ± SEM. Intracellular cAMP was assayed 1 min after the addition of FMLP to the cells. 

Intracellular ATP levels.

The results for intracellular ATP levels are shown in Table 4. Coincubation of neutrophils for 30 min at 37°C with 1-hp at concentrations of up to 12.5 μM did not significantly affect intracellular ATP levels in unstimulated neutrophils.

TABLE 4.

Intracellular ATP levels in neutrophils treated with varying concentrations (1.55 to 12.5 μM) of 1-hp^a

System	Intracellular ATP (nmol/107 cells)
Neutrophils only	23.9 ± 1.1
Neutrophils + 1.55 μM 1-hp	23.8 ± 1.3
Neutrophils + 3.1 μM 1-hp	23.1 ± 1.4
Neutrophils + 6.25 μM 1-hp	22.8 ± 1.2
Neutrophils + 12.5 μM 1-hp	22.9 ± 1.4

^aThe results of 10 experiments are expressed as the mean values ± SEM. 

cAMP-dependent PKA.

Coincubation of PKA with 1-hp had no statistically significant effects on enzyme activity. The activity of PKA coincubated with 12.5 μM 1-hp was 91% ± 4% of that of the corresponding pigment-free control system (data from five experiments).

DISCUSSION

In the present study, treatment of human neutrophils with the P. aeruginosa-derived pigment 1-hp was found to potentiate the release of the primary granule enzymes elastase and MPO, following exposure of these cells to FMLP. Importantly, these data, which are essentially confirmatory (29, 30), were obtained by using concentrations of the pigment which are well within the range reported to occur in the sputa of cystic fibrosis patients colonized with this intransigent microbial pathogen (26). These potentially harmful proinflammatory interactions of 1-hp with neutrophils could not be ascribed to nonspecific cytotoxicity, since ATP levels, a sensitive indicator of cellular damage, were similar in both control and pigment-treated cells. This observation indicated that 1-hp may potentiate biochemical mechanisms which are involved in the activation of neutrophil degranulation or, alternatively, inhibit those which mediate down-regulation of this response. Since degranulation by activated neutrophils is a Ca²⁺-dependent process (16, 18, 23), biochemical processes which mediate increases in the cytosolic concentrations of this cation, as well as those which restore Ca²⁺ homeostasis, were identified as possible targets of 1-hp.

Data from fura-2-based experiments demonstrated that the abruptly occurring increase in cytosolic Ca²⁺ in FMLP-stimulated neutrophils, a response which is due to the release of the cation from intracellular stores (2, 11), was unaffected by 1-hp. These results indicate that 1-hp does not affect the FMLP–receptor–G-protein interactions which lead to activation of phospholipase C (19) nor does it influence the interactions of inositol triphosphate with Ca²⁺-mobilizing receptors located on specialized, intracellular cation storage vesicles (28). Although 1-hp did not affect the peak fura-2 responses of FMLP-activated neutrophils, the rate of decline to basal fluorescence levels was decreased in pigment-treated cells, an observation which is indicative of either a reduction in the efficiency of clearance of cytosolic Ca²⁺ or enhancement of influx of the cation. To identify which, if any, of these was influenced by 1-hp, radiometric procedures were used to monitor fluxes of Ca²⁺ and to distinguish between net efflux and influx of the cation in control and 1-hp-treated, FMLP-activated neutrophils (2).

Activation of neutrophils equilibrated and maintained in cell suspension medium containing ⁴⁵Ca²⁺ was accompanied by an abrupt decrease and gradual recovery in cellular ⁴⁵Ca²⁺, events which appeared to correspond with initial efflux and delayed influx of the cation. Although the type of radiometric procedure used for this initial series of experiments was unable to distinguish between net efflux and influx of Ca²⁺, the results suggested that pretreatment of neutrophils with 1-hp reduces the extent of efflux, without affecting influx of the cation, resulting in intracellular concentrations of Ca²⁺ which are higher than prestimulation values. This observation suggests that exposure of neutrophils to 1-hp not only prolongs the elevation in cytosolic Ca²⁺ levels in stimulated neutrophils, leading to exaggerated proinflammatory activity of these cells, but may also result in Ca²⁺ overload, leading to hyperreactivity of the cells on restimulation with Ca²⁺-mobilizing stimuli.

In the system designed to measure net efflux, exposure of neutrophils, which had been preloaded with ⁴⁵Ca²⁺, to FMLP resulted in a rapid efflux of the cation, an observation which is in agreement with previous reports (10, 21). Efflux of the cation occurred abruptly, coinciding with the peak increase in cytosolic Ca²⁺, and terminated about 30 s after the addition of FMLP. Treatment of neutrophils with 1-hp resulted in a dose-related reduction in efflux of Ca²⁺ from FMLP-activated neutrophils, indicating interference with plasma membrane cation extrusion systems.

Two types of Ca²⁺ efflux systems have been described for human neutrophils. The first of these is a high-capacity, low-affinity Na⁺-Ca²⁺ exchanger (34), the existence of which is uncertain in neutrophils (11, 22, 38). In the present study, we failed to demonstrate influx of Na⁺ into FMLP-activated neutrophils coincident with efflux of Ca²⁺, an observation which does not support the involvement of a Na⁺-Ca²⁺ exchanger in Ca²⁺ efflux in FMLP-activated neutrophils. The second type of Ca²⁺ efflux system is a thapsigargin-insensitive Ca²⁺-ATPase modulated by calmodulin, which shifts the pump to a higher-affinity state for Ca²⁺, resulting in enhanced maximal velocity (17). This system, which is apparently the major Ca²⁺ efflux system operative in human neutrophils (17), is the probable target of 1-hp.

During the brief period of efflux of cytosolic Ca²⁺ from FMLP-activated neutrophils, there was no discernible net influx of the cation. Influx was evident only after completion of efflux, being detected at around 30 to 60 s after the addition of FMLP to neutrophils. As reported previously, the observed influx was initially slow, accelerating at around 2 to 3 min and terminating at 5 min. This delayed influx of Ca²⁺ into FMLP-activated neutrophils is characteristic of a store-operated influx, which is operative in many cell types and is necessary for refilling of stores (8). Treatment of neutrophils with 1-hp did not affect either the rate or the extent of influx of Ca²⁺ into FMLP-activated neutrophils, demonstrating the insensitivity of store-operated influx of the cation to the pigment.

Interestingly, influx of Ca²⁺ into FMLP-activated neutrophils was maximal at a time when fura-2 fluorescence had subsided to around baseline levels. Although this observation may support the existence of a privileged store-filling mechanism by which incoming cation bypasses the cytosol (8, 37), it is more likely to reflect the efficiency of the endomembrane Ca²⁺-ATPase, a thapsigargin-sensitive, cAMP-dependent PKA-modulated cation pump which rapidly sequesters Ca²⁺ into storage vesicles (31, 35). Several lines of evidence suggest that neither the activation nor the activity of the endomembrane Ca²⁺-ATPase is influenced by 1-hp. Firstly, the increase in neutrophil cAMP levels, which accompanies activation of these cells with FMLP (1, 33) and which is probably required for activation of the endomembrane Ca²⁺-ATPase and down-regulation of the proinflammatory activities of activated neutrophils (3), was unaffected by 1-hp, as was the activity of purified PKA in a cell-free assay system. Secondly, although the rate of decline in the fura-2 fluorescence responses of FMLP-activated neutrophils was lower in 1-hp-treated cells than in control cells, a return to basal fluorescence was also observed in pigment-treated cells, indicating that the activity of the endomembrane Ca²⁺-ATPase was intact.

In conclusion, these observations demonstrate that the P. aeruginosa-derived pigment 1-hp exerts its proinflammatory actions on human neutrophils by acting as an antagonist of the plasma membrane Ca²⁺-ATPase. Although the exact molecular mechanism of these antagonistic interactions of the pigment with this Ca²⁺-efflux system remains to be established, the resultant prolongation of the increment in cytosolic Ca²⁺ in activated neutrophils clearly results in hyperactivation of these cells. If operative in vivo, these proinflammatory interactions between 1-hp and neutrophils in the bronchial tree are likely to result in “innocent bystander” injury to lung tissue.