Vol. 60, No. 6
INFEcTION AND IMMUNITY, June 1992, p. 2536-2540
0019-9567/92/062536-05$02.00/0 Copyright © 1992, American Society for Microbiology
Leukotriene B4 w-Oxidation by Human Polymorphonuclear Leukocytes Is Inhibited by Pyocyanin, a Phenazine Derivative Produced by Pseudomonas aeruginosa MICHAEL MULLER* AND TANIA C. SORRELL
Centre for Infectious Diseases and Microbiology, Westmead Hospital, University of Sydney, Westmead, New South Wales 2145, Australia Received 31 December 1991/Accepted 2 April 1992
Human polymorphonuclear leukocytes (PMNL) metabolize the potent chemotaxin leukotriene B4 (LTB4) by 0)-oxidation to 20-hydroxy-LTB4 and 20-carboxy-LTB4. The ability of unstimulated human PMNL to metabolize exogenous LTB4 was found to be inhibited by pyocyanin, a phenazine derivative produced by Pseudomonas aeruginosa, in a dose-dependent manner. 1-Hydroxyphenazine (1-OHP), a metabolite of pyocyanin, was not inhibitory under identical conditions. The initial enzymic step in the conversion of LTB4 is catalyzed by an NADPH-dependent cytochrome, P-450. Reduction of the phenazine derivatives by NADPH was measured spectrophotometrically. Pyocyanin was reduced by NADPH in vitro in a pH-dependent manner, while 1-OHP was poorly or negligibly reduced under similar conditions. Formation of NADP+ was 20.3 + 1.8 nmol min-' for pyocyanin (10 ,uM) at pH 5.5, compared with 0.6 + 0.2 nmol min-' for 1-OHP (10 ,uM), while at pH 7.5 a value of 2.2 ± 1.3 nmol min-' was obtained for pyocyanin, with no detectable activity for 1-OHP. This indicates that inhibition of LTB4 w-hydroxylase activity by pyocyanin might be achieved by competition for NADPH. Incorporation of exogenous 5-hydroxyeicosatetraenoic acid by PMNL into lipid pools was not affected by either phenazine derivative. The ability of bacterial pyocyanin to limit the w-oxidation of LTB4 may have important implications for PMNL LTB4 receptor status and chemotaxis in vivo.
nin but not 1-OHP in unstimulated human PMNL, whereas 5-HETE metabolism remains unaffected.
Stimulation of human polymorphonuclear leukocytes (PMNL) by natural or artificial stimuli elicits production of leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) via the 5-lipoxygenase pathway. These eicosanoids exhibit a number of important biological functions and exert their effects at low concentrations. LTB4 induces human PMNL aggregation, degranulation, adherence to endothelial cell membranes, and chemotaxis, in addition to other functions (3). The biological activity of LTB4 is regulated in human PMNL by w-hydroxylation to the less active but still chemotactic compound 20-hydroxy-LTB4. The enzyme involved has been shown to be a membrane-bound, NADPH-dependent cytochrome designated P-450LTB. (4, 22). In microsomes of human origin, subsequent metabolism and inactivation of 20-hydroxy-LTB4 have been demonstrated to occur by further action of P-450LTBO (23) or by action of NAD+-dependent dehydrogenases to yield 20carboxy-LTB4 (9, 25). 5-HETE has been reported to be processed by unstimulated PMNL by incorporation into triglyceride and phospholipid (19, 24), although w-oxidation of 5-HETE has also been described (20). Pseudomonas aeruginosa, an opportunistic bacterium which is associated with chronic colonization of the lungs of patients with cystic fibrosis, produces substantial quantities of the phenazine derivative pyocyanin (1-hydroxy-5-methylphenazinium hydroxide inner salt). The effects of this derivative and 1-hydroxyphenazine (1-OHP), a metabolite of pyocyanin, on the biosynthesis of LTB4 and 5-HETE by human PMNL have been described by us previously (16). We now present observations that indicate that metabolism of LTB4 to its -oxidation products is inhibited by pyocya-
*
MATERIALS AND METHODS
Preparation of PMNL. Normal PMNL were obtained from the blood of healthy volunteers. Briefly, PMNL were prepared from 30 ml of anticoagulated venous blood by centrifugation at 250 x g for 45 min through 20 ml of Mono-Poly Resolving medium (Flow Laboratories, Sydney, Australia). The PMNL layer was removed and washed with calciumand magnesium-free Hanks balanced salt solution. Contaminating erythrocytes were removed by hypotonic lysis. PMNL (95% purity or greater) were washed, suspended in complete Hanks balanced salt solution containing calcium and magnesium, and used at a concentration of 5 x 106 cells per ml for all experiments. [5,6,8,9,11,12,14,15(n)-3H]-LTB4 ([3H]LTB4) and 5(S)-hydroxy-[5,6,8,9, 11,12,14,15(n)-3H]eicosatetraenoic acid ([3H]5-HETE) were obtained from Amersham (Sydney, Australia), and 20-[14,15-3H(N)]-hydroxy LTB4 was obtained from DuPont (Sydney, Australia). Preparation of phenazine derivatives. Pyocyanin was prepared by a modification of the photochemical method of Knight et al. (12). Phenazine methosulfate (Sigma) was made up in 0.01 M Tris-HCl buffer (pH 7.4) in a Pyrex container exposed to fluorescent light for 10 h. The solution was extracted twice with 1 volume of chloroform. The pooled extracts were evaporated to dryness under vacuum. This residue was suspended in chloroform and acidified with 1 volume of 0.1 M HCl. The aqueous phase was washed twice with 1 volume of chloroform. Sufficient 0.5 M NaOH was added to bring the solution to neutrality, and the aqueous solution was extracted twice with 1 volume of chloroform. The pooled organic extracts were reduced to
Corresponding author. 2536
VOL. 60, 1992
PHENAZINES ALTER LTB4 METABOLISM IN NEUTROPHILS
dryness under vacuum and washed with 3 volumes of hexane. The residue was suspended in chloroform, and sufficient hexane was added to precipitate the pyocyanin. 1-OHP was prepared by a modification of the method of Armstrong et al. (1). Pyocyanin was added to a 1% solution of NaOH and allowed to stand in the dark for 3 h. The solution was acidified with 1 M HCl until a yellow solution was obtained. The crude 1-OHP was extracted twice into 1 volume of chloroform, and the pooled extracts were evaporated to dryness under vacuum. Final purification of pyocyanin and 1-OHP was performed by thin-layer chromatography on silica gel G with chloroform-methanol (9:1, vol/vol). The identity of each derivative was confirmed by comparison of its UV spectrum and extinction coefficient with established values (27). Before use, the purity of each derivative was checked by thin-layer chromatography as described above. Determination of cell viability. Prior to conducting experiments, we investigated the effect of high doses of either pyocyanin or 1-OHP on PMNL viability by incubating 5 x 106 PMNL per ml in 0, 50, 100, and 200 ,uM solutions of either derivative in Hanks balanced salt solution for 30 min at 37°C. Viability was assessed by trypan blue dye exclusion. Metabolic studies. Time course experiments to determine how PMNL metabolize [3H]LTB4 and [3H]5-HETE were performed by incubating 25 nCi of either compound with 5 x 106 PMNL per ml at 37°C for 60 min. Aliquots were withdrawn at intervals, and the metabolites were extracted. Dose-response experiments were then performed to determine the effect of pyocyanin or 1-OHP on the metabolism of [3H]LTB4 and [3H]5-HETE. PMNL were preincubated with the appropriate phenazine derivative for 20 min at 37°C with shaking. Twenty-five nanocuries of either [3H]LTB4 or [3H]5-HETE was then added in 10 ,ul of complete Hanks balanced salt solution, and the suspension was incubated for 30 ([3H]LTB4) or 2 ([3H]5-HETE) min. Extraction and separation of metabolites. All reactions were terminated by addition of 1 volume of methanol. After extraction by the method of Bligh and Dyer (2), arachidonic acid metabolites were suspended in chloroform-methanol (9:1, vol/vol) and separated by thin-layer chromatography on silica gel G by using a solvent system consisting of chloroform-methanol-acetic acid-water (90:8:1:0.8, vol/vol/vol/vol) (18). Gel bands 1 cm wide were removed from the plate and counted in a Packard scintillation counter. LTB4 and its derivatives obtained from incubation with PMNL were identified by comigration with authentic standards of [3H]LTB4, LTB4, [ H]20-OH-LTB4 and 20-OH-LTB4 on thin-layer chromatography by using the system described above. The identity of each unlabelled compound was further checked by elution from the appropriate gel band with methanol and determination of its UV spectrum. In each case, the conjugated triene structure with absorption maxima characteristic of LTB4 or its derivatives was found. Spectrophotometric assays. The absorption spectrum of 0.1 mM NADPH in 0.1 M Tris-HCl buffer was determined alone and in the presence of 10 p,M pyocyanin or 1-OHP for different pH values at 22°C over time. RESULTS PMNL viability. After incubation with 200 p,M pyocyanin or 1-OHP for 30 min at 370C, 98.3% + 6.9% and 97.8% + 14.3% of PMNL, respectively, were found to be viable, as judged by the trypan blue dye exclusion test. Eicosanoid metabolism. We investigated the metabolism
2537
100
A
o.-o LTB4 D--Oxidation
80
Products 0
0 0
._
60
o
40
020 ._
10
20
30
40
50
60
100 t
B
o-o 5-HETE --
80
-o
Triglyceride
0
.0
0
60
0 0
'II
40~
-I o
I~~~~~~~~~~~~~
20
0
10
20
30
40
50
60
Time (min) FIG. 1. Time course for metabolism of LTB4 and 5-HETE by PMNL. Metabolism by 5 x 106 PMNL per ml over 30 min at 37°C in complete Hanks balanced salt solution of [3H]LTB4 and formation of total b-oxidation products (A) and metabolism of [3H]5-HETE and formation of labelled triglyceride (B). The results are expressed as percentages of the total radioactivity recovered (mean ± standard deviation of three experiments).
and distribution into lipid classes of labelled LTB4 and 5-HETE by untreated PMNL and compared the results with those obtained with pyocyanin- or 1-OHP-treated cells. As shown in Fig. 1A, LTB4 was converted predominantly to o-oxidation products. We found that 5% or less of the label was incorporated into neutral lipid and phospholipid at any time point between 0 and 60 min, in agreement with the work of others (21) (data not shown). Under identical conditions, 5-HETE was metabolized relatively rapidly. A time of 18 min was required to reduce the initial amount of LTB4 by 50%, whereas a similar reduction in 5-HETE required only 2.5 min. 5-HETE was incorporated predominantly into neutral lipid, with negligible incorporation into phospholipid or production of b-oxidation products. Thin-layer chromatography of the neutral lipid fractions revealed that 5-HETE was incorporated into triglyceride (Fig. 1B). While this was in agreement with previously published results, the low incorporation into phospholipid was contrary to previous reports (20, 24). Treatment with phenazines. Pretreatment with increasing concentrations of pyocyanin (12.5 to 200 pM) resulted in a dose-dependent decrease in the formation of w-oxidation products of LTB4 (Fig. 2). The dose of pyocyanin required to achieve 50% inhibition was calculated to be 87.5 p,M. The major product recovered after incubation with pyocyanin comigrated with authentic LTB4 on thin-layer chromatogra-
2538
INFECT. IMMUN.
MULLER AND SORRELL
TABLE 1. Effect of pH on NADP+ formationa
80
pH 0
(6 co
0-0
cc
o-0o Oxidation Products
"s %L4T
0
I-.
LT B4
3.5 4.5 5.5 6.5 7.5 8.5 9.5
Mean NADP+ formation (nmol
Pyocyanin
29.2 27.6 20.3 4.5 2.2 1.5 1.3
± ± ± ± ± ± ±
1.2 1.1 1.8 0.6 1.3 0.1 0.0
min-') ± SD 1-OHP
2.3 0.8 0.6 0.2 0.0 0.0 0.0
± ± ± ± ± ± ±
0.7 0.3 0.2 0.1 0.0 0.0 0.0
-8 a The effect of pH on the rate of formation of NADP+ by 10 AM pyocyanin or 1-OHP from 0.1 mM NADPH in 0.1 M Tris-HCl buffer at 22°C was studied. 200 The results of three experiments are shown.
-------
0
25
50
75
100
125
150
175
Pyocyanin (pM) FIG. 2. Effect of pyocyanin concentration on metabolism of [3H]LTB4 and formation of w-oxidation products after incubation for 30 min with 5 x 106 PMNL per ml. The results are expressed as percentages of the total radioactivity recovered (mean ± standard deviation of three experiments).
phy and not with o-oxidation metabolites. Inhibition of LTB4 metabolism was not observed in PMNL treated with 1-OHP (Fig. 3). [3H]5-HETE metabolism was not altered by treatment with either pyocyanin or 1-OHP (data not shown). Under the conditions employed for these studies, no w-oxidation of 5-HETE was observed. Spectrophotometric studies. To determine the mechanism responsible for the differential inhibition observed with the two phenazine derivatives, we measured the rates of reduction of pyocyanin and 1-OHP by NADPH spectrophotometrically. We found a significant difference in the rate of reaction between the two derivatives. Pyocyanin (10 ,uM) was reduced at all pH values at a significantly greater rate than 1-OHP and in a pH-dependent manner (Table 1). At pHs of .4.5, negligible reduction of 1-OHP (10 p,M) occurred over 60 min. DISCUSSION In a previous study, we established that pyocyanin and 1-OHP inhibited production of the 5-lipoxygenase products
0
80
0
0-o Pyocyanin
-
-
0 c
0
0.
60
o---o 1-OHP
40 20
0
25
50
75
100
125
150
175
200
Concentration (pM) FIG. 3. Comparison of the effects of pyocyanin and 1-OHP on inhibition of LTB4 metabolism. [3H]LTB4 was incubated with 5 x 106 PMNL per ml for 30 min in the presence of either pyocyanin or 1-OHP. The results are expressed as percentages of the control (mean ± standard deviation of three experiments).
LTB4 and 5-HETE from stimulated human PMNL at concentrations of 50 jxM or less. In addition, we observed a decline in the production of LTB4 w-oxidation products, presumably owing to a reduction in the amount of the LTB4 substrate available (16). In this study, we investigated the effects of phenazine derivatives on the metabolism of exogenous [3H]LTB4 and [3H]5-HETE by PMNL. Exposure of unstimulated PMNL to clinically relevant concentrations (28) of either pyocyanin or 1-OHP resulted in dose-dependent inhibition of LTB4 metabolism only in the case of pyocyanin, while 1-OHP was without effect. As the dominant product recovered after incubation of PMNL with exogenous LTB4 and pyocyanin was the parent compound and not intermediates of the w-oxidation pathway, we conclude that the initial step, conversion of LTB4 to 20-OHLTB4, is the site of action of pyocyanin. In investigating the reason for the discrepancy between the actions of pyocyanin and 1-OHP, we examined their abilities to oxidize NADPH at various pH values. PMNL degranulation, a process promoted by LTB4 (3), is thought to induce localized intracellular pH changes in PMNL. Although precise local pH values are not known, a pH of 5.0 is considered possible (11). P-450LTB. has been shown to be a membrane-bound, NADPH-dependent system (22). Substantial depletion of intracellular NADPH by pyocyanin has been reported to occur in human PMNL (15), and we have established in the present study that pyocyanin can initiate NADPH oxidation in a pH-dependent manner, whereas 1-OHP does so only poorly. We conclude that inhibition of P-450LThB. by pyocyanin is achieved by causing the substrate, NADPH, to become limiting and propose the mechanism illustrated in Fig. 4 to account for our observations. That 1-OHP is ineffective as an inhibitor of LTB4 metabolism is most likely due to its poor ability to oxidize NADPH. The interaction of reduced pyocyanin with molecular oxygen can generate the oxygen free radical superoxide (10). It has been proposed that free radicals generated by phenazine derivatives can inactivate dehydrogenase systems in vitro (5), and we have found that pyocyanin and 1-OHP, using NADH as an electron donor, alter lactate dehydrogenase activity (17). However, the possible inactivation of P-45OLTB. by this mechanism is unlikely, as it has been previously demonstrated in unstimulated PMNL that pyocyanin concentrations of up to 200 ,uM did not lead to formation of superoxide but did cause significant depletion of NADPH (15). Metabolism of 5-HETE by PMNL treated with pyocyanin or 1-OHP was not affected, indicating that the acyltransferase(s) involved in esterifying 5-HETE into neutral lipid is
PHENAZINES ALTER LTB4 METABOLISM IN NEUTROPHILS
VOL. 60, 1992
PYo
NADPH
LTB4 --,
P-450 PYOH2
NADP
#--'
20-Hydroxy-LTB4
20-Carboxy- LTB4 FIG. 4. Proposed mechanism for inhibition of LTB4 metabolism
by pyocyanin (PYO).
not phenazine sensitive. Although c-oxidation of 5-HETE has been reported to occur at high substrate concentrations
(20), under the conditions employed in this study we were unable to detect w-oxidized metabolites of 5-HETE and, hence, could not evaluate the influence of phenazine derivatives on this activity. While LTB4 is not the only chemotaxin present at infection sites, it is a significant endogenous chemotaxin when compared on a mole-for-mole basis with other substances (7). It has been demonstrated that LTB4 is the major PMNL chemotaxin produced by human alveolar macrophages (14). Possession by P. aeruginosa of a mechanism for preventing inactivation of LTB4 by human PMNL appears to be inconsistent with long-term survival of the organism, since increased chemotaxis of PMNL and macrophages in response to LTB4 should result in increased phagocytosis and bacterial killing at the infection site. However, it has been demonstrated that prior exposure of human PMNL to LTB4 leads to loss of their chemotactic responsiveness with respect to LTB4 (6). This loss has been attributed to down regulation of high-affinity LTB4 receptors on the cell surface (8, 26). As PMNL are the only cells known to inactivate LTB4 effectively by c-oxidation (13), the inability of PMNL exposed to pyocyanin to inactivate this compound may result in down regulation of PMNL LTB4 receptors in vivo and may reduce LTB4-mediated chemotaxis. Thus, an advantage may be conferred upon the pathogen for survival within the host. PMNL infiltration into infection sites in response to other chemotaxins could still occur, and these substances may then become more important than LTB4 as the principal chemotaxins. We conclude that pyocyanin appears to play an important role in altering the synthesis and subsequent metabolism of LTB4 by human PMNL. As LTB4 is considered to be a major mediator of inflammation, we conclude that bacterial pyocyanin may have a significant role as a virulence factor in this process. Thus, pyocyanin may contribute to the persistence of P. aeruginosa in chronic disease, particularly in lung infections such as cystic fibrosis and bronchiectasis.
2539
ACKNOWLEDGMENT This work was supported by Merck, Sharp and Dohme (Australia), Proprietary Limited. REFERENCES 1. Armstrong, A. V., D. E. S. Stewart-Tuli, and J. S. Roberts. 1971. Characterisation of the Pseudomonas aeruginosa factor that inhibits mouse-liver mitochondrial respiration. J. Med. Microbiol. 4:249-262. 2. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911917. 3. Bray, M. A. 1983. The pharmacology and pathophysiology of leukotriene B4. Br. Med. Bull. 39:249-254. 4. Brom, J., W. Konig, M. Stuining, M. Raulf, and M. Koller. 1987. Characterization of leukotriene B4-omega-hydroxylase activity within human polymorphonuclear granulocytes. Scand. J. Immunol. 25:283-294. 5. Davis, G., and P. J. Thornalley. 1983. Free radical production from the aerobic oxidation of reduced pyridine nucleotides catalysed by phenazine derivatives. Biochim. Biophys. Acta 724:456-464. 6. Goetzl, E. J., J. M. Boeynaems, J. A. Oates, and W. C. Hubbard. 1981. Stimulus-specificity of the chemotactic deactivation of human neutrophils by lipoxygenase products of arachidonic acid. Prostaglandins 22:279-288. 7. Goetzl, E. J., and W. C. Pickett. 1980. The human PMN leukocyte chemotactic activity of complex hydroxyeicosatetraenoic acids (HETEs). J. Immunol. 125:1789-1791. 8. Goldman, D. W., and E. J. Goetzl. 1984. Heterogeneity of human polymorphonuclear leukocyte receptors for leukotriene B4. J. Exp. Med. 159:1027-1041. 9. Gotoh, Y., H. Sumimoto, and S. Minakami. 1989. Purification and characterization of 20-hydroxy-leukotriene B4 dehydrogenase in human neutrophils. Eur. J. Biochem. 179:315-321. 10. Hassan, H. M., and I. Fridovich. 1980. Mechanism of the antibiotic action of pyocyanin. J. Bacteriol. 141:156-163. 11. Henson, P. M., J. E. Henson, C. Fittschen, G. Kimani, D. L. Bratton, and W. H. Riches. 1988. Phagocytic cells: degranulation and secretion, p. 363-390. In J. I. Gallin, I. M. Goldstein, and R. Snyderman (ed.), Inflammation: basic principles and clinical correlates. Raven Press, New York. 12. Knight, M., P. E. Hartman, Z. Hartman, and V. M. Young. 1979. A new method of preparation of pyocyanin and demonstration of an unusual bacterial sensitivity. Anal. Biochem. 95:19-23. 13. Lewis, R. A., and K. F. Austen. 1988. Leukotrienes, p. 121-128. In J. I. Gallin, I. M. Goldstein, and R. Snyderman (ed.), Inflammation: basic principles and clinical correlates. Raven Press, New York. 14. Martin, T. R., G. Raugi, T. L. Merritt, and W. R. Henderson. 1987. Relative contribution of leukotriene B4 to the neutrophil chemotactic activity produced by the resident human alveolar macrophage. J. Clin. Invest. 80:1114-1124. 15. Muller, P. K., K. Krohn, and P. F. Muhlradt. 1989. Effects of pyocyanine, a phenazine dye from Pseudomonas aeruginosa, on oxidative burst and bacterial killing in human neutrophils. Infect. Immun. 57:2591-2596. 16. Muller, M., and T. C. Sorrell. 1991. Production of leukotriene B4 and 5-hydroxyeicosatetraenoic acid by human neutrophils is inhibited by Pseudomonas aeruginosa phenazine derivatives. Infect. Immun. 59:3316-3318. 17. Muller, M., and T. C. Sorrell. 1991. Effect of pyocyanin and 1-hydroxphenazine on the assay of lactate dehydrogenase activity. J. Infect. Dis. 164:610-611. 18. Nugteren, D. H., and E. Hazelhof. 1973. Isolation and properties of intermediates in prostaglandin biosynthesis. Biochim. Biophys. Acta 326:448-461. 19. O'Flaherty, J. T. 1987. Phospholipid metabolism and stimulusresponse coupling. Biochem. Pharmacol. 36:407-412. 20. O'Flaherty, J. T., R. L. Wykle, J. Redman, M. Samuel, and M. Thomas. 1986. Metabolism of 5-hydroxyeicosatetraenoate by human neutrophils: production of a novel w-oxidized derivative.
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J. Immunol. 137:3277-3283. 21. Shak, S., and I. M. Goldstein. 1984. w-Oxidation is the major pathway for the catabolism of leukotriene B4 in human polymorphonuclear leukocytes. J. Biol. Chem. 259:10181-10187. 22. Shak, S., and I. M. Goldstein. 1985. Leukotriene B4 W-hydroxylase in human polymorphonuclear leukocytes: partial purification and identification as a cytochrome P-450. J. Clin. Invest. 76:1218-1228. 23. Soberman, R. J., J. P. Sutyak, R. T. Okita, D. F. Wendelborn, L. J. Roberts U, and K F. Austen. 1988. The identification and formation of 20-aldehyde leukotriene B4. J. Biol. Chem. 263: 7996-8002. 24. Stenson, W. F., and C. W. Parker. 1979. Metabolism of arachidonic acid in ionophore-stimulated neutrophils. J. Clin. Invest. 64:1457-1465. 25. Sumimoto, H., and S. Minakami. 1990. Oxidation of 20-hydrox-
INFECT. IMMUN.
yleukotriene B4 to 20-carboxyleukotriene B4 by human neutrophil microsomes. J. Biol. Chem. 265:4348-4353. 26. Vitkauskas, G., H. J. Showell, and E. L. Becker. 1980. Specific binding of synthetic chemotactic peptides to rabbit peritoneal neutrophils: effects on dissociability of bound peptide, receptor activity, and subsequent biologic responsiveness (deactivation). Mol. Immunol. 17:171-180. 27. Watson, D., J. MacDermot, R. Wilson, P. J. Cole, and G. W. Taylor. 1986. Purification and structural analysis of pyocyanin and 1-hydroxyphenazine. Eur. J. Biochem. 159:309-313. 28. Wilson, R., D. A. Sykes, D. Watson, A. Rutman, G. W. Taylor, and P. J. Cole. 1988. Measurement of Pseudomonas aeruginosa phenazine pigments in sputum and assessment of their contribution to sputum sol toxicity for respiratory epithelium. Infect. Immun. 56:2515-2517.
INFEcTION AND IMMUNITY, June 1992, p. 2536-2540
0019-9567/92/062536-05$02.00/0 Copyright © 1992, American Society for Microbiology
Leukotriene B4 w-Oxidation by Human Polymorphonuclear Leukocytes Is Inhibited by Pyocyanin, a Phenazine Derivative Produced by Pseudomonas aeruginosa MICHAEL MULLER* AND TANIA C. SORRELL
Centre for Infectious Diseases and Microbiology, Westmead Hospital, University of Sydney, Westmead, New South Wales 2145, Australia Received 31 December 1991/Accepted 2 April 1992
Human polymorphonuclear leukocytes (PMNL) metabolize the potent chemotaxin leukotriene B4 (LTB4) by 0)-oxidation to 20-hydroxy-LTB4 and 20-carboxy-LTB4. The ability of unstimulated human PMNL to metabolize exogenous LTB4 was found to be inhibited by pyocyanin, a phenazine derivative produced by Pseudomonas aeruginosa, in a dose-dependent manner. 1-Hydroxyphenazine (1-OHP), a metabolite of pyocyanin, was not inhibitory under identical conditions. The initial enzymic step in the conversion of LTB4 is catalyzed by an NADPH-dependent cytochrome, P-450. Reduction of the phenazine derivatives by NADPH was measured spectrophotometrically. Pyocyanin was reduced by NADPH in vitro in a pH-dependent manner, while 1-OHP was poorly or negligibly reduced under similar conditions. Formation of NADP+ was 20.3 + 1.8 nmol min-' for pyocyanin (10 ,uM) at pH 5.5, compared with 0.6 + 0.2 nmol min-' for 1-OHP (10 ,uM), while at pH 7.5 a value of 2.2 ± 1.3 nmol min-' was obtained for pyocyanin, with no detectable activity for 1-OHP. This indicates that inhibition of LTB4 w-hydroxylase activity by pyocyanin might be achieved by competition for NADPH. Incorporation of exogenous 5-hydroxyeicosatetraenoic acid by PMNL into lipid pools was not affected by either phenazine derivative. The ability of bacterial pyocyanin to limit the w-oxidation of LTB4 may have important implications for PMNL LTB4 receptor status and chemotaxis in vivo.
nin but not 1-OHP in unstimulated human PMNL, whereas 5-HETE metabolism remains unaffected.
Stimulation of human polymorphonuclear leukocytes (PMNL) by natural or artificial stimuli elicits production of leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) via the 5-lipoxygenase pathway. These eicosanoids exhibit a number of important biological functions and exert their effects at low concentrations. LTB4 induces human PMNL aggregation, degranulation, adherence to endothelial cell membranes, and chemotaxis, in addition to other functions (3). The biological activity of LTB4 is regulated in human PMNL by w-hydroxylation to the less active but still chemotactic compound 20-hydroxy-LTB4. The enzyme involved has been shown to be a membrane-bound, NADPH-dependent cytochrome designated P-450LTB. (4, 22). In microsomes of human origin, subsequent metabolism and inactivation of 20-hydroxy-LTB4 have been demonstrated to occur by further action of P-450LTBO (23) or by action of NAD+-dependent dehydrogenases to yield 20carboxy-LTB4 (9, 25). 5-HETE has been reported to be processed by unstimulated PMNL by incorporation into triglyceride and phospholipid (19, 24), although w-oxidation of 5-HETE has also been described (20). Pseudomonas aeruginosa, an opportunistic bacterium which is associated with chronic colonization of the lungs of patients with cystic fibrosis, produces substantial quantities of the phenazine derivative pyocyanin (1-hydroxy-5-methylphenazinium hydroxide inner salt). The effects of this derivative and 1-hydroxyphenazine (1-OHP), a metabolite of pyocyanin, on the biosynthesis of LTB4 and 5-HETE by human PMNL have been described by us previously (16). We now present observations that indicate that metabolism of LTB4 to its -oxidation products is inhibited by pyocya-
*
MATERIALS AND METHODS
Preparation of PMNL. Normal PMNL were obtained from the blood of healthy volunteers. Briefly, PMNL were prepared from 30 ml of anticoagulated venous blood by centrifugation at 250 x g for 45 min through 20 ml of Mono-Poly Resolving medium (Flow Laboratories, Sydney, Australia). The PMNL layer was removed and washed with calciumand magnesium-free Hanks balanced salt solution. Contaminating erythrocytes were removed by hypotonic lysis. PMNL (95% purity or greater) were washed, suspended in complete Hanks balanced salt solution containing calcium and magnesium, and used at a concentration of 5 x 106 cells per ml for all experiments. [5,6,8,9,11,12,14,15(n)-3H]-LTB4 ([3H]LTB4) and 5(S)-hydroxy-[5,6,8,9, 11,12,14,15(n)-3H]eicosatetraenoic acid ([3H]5-HETE) were obtained from Amersham (Sydney, Australia), and 20-[14,15-3H(N)]-hydroxy LTB4 was obtained from DuPont (Sydney, Australia). Preparation of phenazine derivatives. Pyocyanin was prepared by a modification of the photochemical method of Knight et al. (12). Phenazine methosulfate (Sigma) was made up in 0.01 M Tris-HCl buffer (pH 7.4) in a Pyrex container exposed to fluorescent light for 10 h. The solution was extracted twice with 1 volume of chloroform. The pooled extracts were evaporated to dryness under vacuum. This residue was suspended in chloroform and acidified with 1 volume of 0.1 M HCl. The aqueous phase was washed twice with 1 volume of chloroform. Sufficient 0.5 M NaOH was added to bring the solution to neutrality, and the aqueous solution was extracted twice with 1 volume of chloroform. The pooled organic extracts were reduced to
Corresponding author. 2536
VOL. 60, 1992
PHENAZINES ALTER LTB4 METABOLISM IN NEUTROPHILS
dryness under vacuum and washed with 3 volumes of hexane. The residue was suspended in chloroform, and sufficient hexane was added to precipitate the pyocyanin. 1-OHP was prepared by a modification of the method of Armstrong et al. (1). Pyocyanin was added to a 1% solution of NaOH and allowed to stand in the dark for 3 h. The solution was acidified with 1 M HCl until a yellow solution was obtained. The crude 1-OHP was extracted twice into 1 volume of chloroform, and the pooled extracts were evaporated to dryness under vacuum. Final purification of pyocyanin and 1-OHP was performed by thin-layer chromatography on silica gel G with chloroform-methanol (9:1, vol/vol). The identity of each derivative was confirmed by comparison of its UV spectrum and extinction coefficient with established values (27). Before use, the purity of each derivative was checked by thin-layer chromatography as described above. Determination of cell viability. Prior to conducting experiments, we investigated the effect of high doses of either pyocyanin or 1-OHP on PMNL viability by incubating 5 x 106 PMNL per ml in 0, 50, 100, and 200 ,uM solutions of either derivative in Hanks balanced salt solution for 30 min at 37°C. Viability was assessed by trypan blue dye exclusion. Metabolic studies. Time course experiments to determine how PMNL metabolize [3H]LTB4 and [3H]5-HETE were performed by incubating 25 nCi of either compound with 5 x 106 PMNL per ml at 37°C for 60 min. Aliquots were withdrawn at intervals, and the metabolites were extracted. Dose-response experiments were then performed to determine the effect of pyocyanin or 1-OHP on the metabolism of [3H]LTB4 and [3H]5-HETE. PMNL were preincubated with the appropriate phenazine derivative for 20 min at 37°C with shaking. Twenty-five nanocuries of either [3H]LTB4 or [3H]5-HETE was then added in 10 ,ul of complete Hanks balanced salt solution, and the suspension was incubated for 30 ([3H]LTB4) or 2 ([3H]5-HETE) min. Extraction and separation of metabolites. All reactions were terminated by addition of 1 volume of methanol. After extraction by the method of Bligh and Dyer (2), arachidonic acid metabolites were suspended in chloroform-methanol (9:1, vol/vol) and separated by thin-layer chromatography on silica gel G by using a solvent system consisting of chloroform-methanol-acetic acid-water (90:8:1:0.8, vol/vol/vol/vol) (18). Gel bands 1 cm wide were removed from the plate and counted in a Packard scintillation counter. LTB4 and its derivatives obtained from incubation with PMNL were identified by comigration with authentic standards of [3H]LTB4, LTB4, [ H]20-OH-LTB4 and 20-OH-LTB4 on thin-layer chromatography by using the system described above. The identity of each unlabelled compound was further checked by elution from the appropriate gel band with methanol and determination of its UV spectrum. In each case, the conjugated triene structure with absorption maxima characteristic of LTB4 or its derivatives was found. Spectrophotometric assays. The absorption spectrum of 0.1 mM NADPH in 0.1 M Tris-HCl buffer was determined alone and in the presence of 10 p,M pyocyanin or 1-OHP for different pH values at 22°C over time. RESULTS PMNL viability. After incubation with 200 p,M pyocyanin or 1-OHP for 30 min at 370C, 98.3% + 6.9% and 97.8% + 14.3% of PMNL, respectively, were found to be viable, as judged by the trypan blue dye exclusion test. Eicosanoid metabolism. We investigated the metabolism
2537
100
A
o.-o LTB4 D--Oxidation
80
Products 0
0 0
._
60
o
40
020 ._
10
20
30
40
50
60
100 t
B
o-o 5-HETE --
80
-o
Triglyceride
0
.0
0
60
0 0
'II
40~
-I o
I~~~~~~~~~~~~~
20
0
10
20
30
40
50
60
Time (min) FIG. 1. Time course for metabolism of LTB4 and 5-HETE by PMNL. Metabolism by 5 x 106 PMNL per ml over 30 min at 37°C in complete Hanks balanced salt solution of [3H]LTB4 and formation of total b-oxidation products (A) and metabolism of [3H]5-HETE and formation of labelled triglyceride (B). The results are expressed as percentages of the total radioactivity recovered (mean ± standard deviation of three experiments).
and distribution into lipid classes of labelled LTB4 and 5-HETE by untreated PMNL and compared the results with those obtained with pyocyanin- or 1-OHP-treated cells. As shown in Fig. 1A, LTB4 was converted predominantly to o-oxidation products. We found that 5% or less of the label was incorporated into neutral lipid and phospholipid at any time point between 0 and 60 min, in agreement with the work of others (21) (data not shown). Under identical conditions, 5-HETE was metabolized relatively rapidly. A time of 18 min was required to reduce the initial amount of LTB4 by 50%, whereas a similar reduction in 5-HETE required only 2.5 min. 5-HETE was incorporated predominantly into neutral lipid, with negligible incorporation into phospholipid or production of b-oxidation products. Thin-layer chromatography of the neutral lipid fractions revealed that 5-HETE was incorporated into triglyceride (Fig. 1B). While this was in agreement with previously published results, the low incorporation into phospholipid was contrary to previous reports (20, 24). Treatment with phenazines. Pretreatment with increasing concentrations of pyocyanin (12.5 to 200 pM) resulted in a dose-dependent decrease in the formation of w-oxidation products of LTB4 (Fig. 2). The dose of pyocyanin required to achieve 50% inhibition was calculated to be 87.5 p,M. The major product recovered after incubation with pyocyanin comigrated with authentic LTB4 on thin-layer chromatogra-
2538
INFECT. IMMUN.
MULLER AND SORRELL
TABLE 1. Effect of pH on NADP+ formationa
80
pH 0
(6 co
0-0
cc
o-0o Oxidation Products
"s %L4T
0
I-.
LT B4
3.5 4.5 5.5 6.5 7.5 8.5 9.5
Mean NADP+ formation (nmol
Pyocyanin
29.2 27.6 20.3 4.5 2.2 1.5 1.3
± ± ± ± ± ± ±
1.2 1.1 1.8 0.6 1.3 0.1 0.0
min-') ± SD 1-OHP
2.3 0.8 0.6 0.2 0.0 0.0 0.0
± ± ± ± ± ± ±
0.7 0.3 0.2 0.1 0.0 0.0 0.0
-8 a The effect of pH on the rate of formation of NADP+ by 10 AM pyocyanin or 1-OHP from 0.1 mM NADPH in 0.1 M Tris-HCl buffer at 22°C was studied. 200 The results of three experiments are shown.
-------
0
25
50
75
100
125
150
175
Pyocyanin (pM) FIG. 2. Effect of pyocyanin concentration on metabolism of [3H]LTB4 and formation of w-oxidation products after incubation for 30 min with 5 x 106 PMNL per ml. The results are expressed as percentages of the total radioactivity recovered (mean ± standard deviation of three experiments).
phy and not with o-oxidation metabolites. Inhibition of LTB4 metabolism was not observed in PMNL treated with 1-OHP (Fig. 3). [3H]5-HETE metabolism was not altered by treatment with either pyocyanin or 1-OHP (data not shown). Under the conditions employed for these studies, no w-oxidation of 5-HETE was observed. Spectrophotometric studies. To determine the mechanism responsible for the differential inhibition observed with the two phenazine derivatives, we measured the rates of reduction of pyocyanin and 1-OHP by NADPH spectrophotometrically. We found a significant difference in the rate of reaction between the two derivatives. Pyocyanin (10 ,uM) was reduced at all pH values at a significantly greater rate than 1-OHP and in a pH-dependent manner (Table 1). At pHs of .4.5, negligible reduction of 1-OHP (10 p,M) occurred over 60 min. DISCUSSION In a previous study, we established that pyocyanin and 1-OHP inhibited production of the 5-lipoxygenase products
0
80
0
0-o Pyocyanin
-
-
0 c
0
0.
60
o---o 1-OHP
40 20
0
25
50
75
100
125
150
175
200
Concentration (pM) FIG. 3. Comparison of the effects of pyocyanin and 1-OHP on inhibition of LTB4 metabolism. [3H]LTB4 was incubated with 5 x 106 PMNL per ml for 30 min in the presence of either pyocyanin or 1-OHP. The results are expressed as percentages of the control (mean ± standard deviation of three experiments).
LTB4 and 5-HETE from stimulated human PMNL at concentrations of 50 jxM or less. In addition, we observed a decline in the production of LTB4 w-oxidation products, presumably owing to a reduction in the amount of the LTB4 substrate available (16). In this study, we investigated the effects of phenazine derivatives on the metabolism of exogenous [3H]LTB4 and [3H]5-HETE by PMNL. Exposure of unstimulated PMNL to clinically relevant concentrations (28) of either pyocyanin or 1-OHP resulted in dose-dependent inhibition of LTB4 metabolism only in the case of pyocyanin, while 1-OHP was without effect. As the dominant product recovered after incubation of PMNL with exogenous LTB4 and pyocyanin was the parent compound and not intermediates of the w-oxidation pathway, we conclude that the initial step, conversion of LTB4 to 20-OHLTB4, is the site of action of pyocyanin. In investigating the reason for the discrepancy between the actions of pyocyanin and 1-OHP, we examined their abilities to oxidize NADPH at various pH values. PMNL degranulation, a process promoted by LTB4 (3), is thought to induce localized intracellular pH changes in PMNL. Although precise local pH values are not known, a pH of 5.0 is considered possible (11). P-450LTB. has been shown to be a membrane-bound, NADPH-dependent system (22). Substantial depletion of intracellular NADPH by pyocyanin has been reported to occur in human PMNL (15), and we have established in the present study that pyocyanin can initiate NADPH oxidation in a pH-dependent manner, whereas 1-OHP does so only poorly. We conclude that inhibition of P-450LThB. by pyocyanin is achieved by causing the substrate, NADPH, to become limiting and propose the mechanism illustrated in Fig. 4 to account for our observations. That 1-OHP is ineffective as an inhibitor of LTB4 metabolism is most likely due to its poor ability to oxidize NADPH. The interaction of reduced pyocyanin with molecular oxygen can generate the oxygen free radical superoxide (10). It has been proposed that free radicals generated by phenazine derivatives can inactivate dehydrogenase systems in vitro (5), and we have found that pyocyanin and 1-OHP, using NADH as an electron donor, alter lactate dehydrogenase activity (17). However, the possible inactivation of P-45OLTB. by this mechanism is unlikely, as it has been previously demonstrated in unstimulated PMNL that pyocyanin concentrations of up to 200 ,uM did not lead to formation of superoxide but did cause significant depletion of NADPH (15). Metabolism of 5-HETE by PMNL treated with pyocyanin or 1-OHP was not affected, indicating that the acyltransferase(s) involved in esterifying 5-HETE into neutral lipid is
PHENAZINES ALTER LTB4 METABOLISM IN NEUTROPHILS
VOL. 60, 1992
PYo
NADPH
LTB4 --,
P-450 PYOH2
NADP
#--'
20-Hydroxy-LTB4
20-Carboxy- LTB4 FIG. 4. Proposed mechanism for inhibition of LTB4 metabolism
by pyocyanin (PYO).
not phenazine sensitive. Although c-oxidation of 5-HETE has been reported to occur at high substrate concentrations
(20), under the conditions employed in this study we were unable to detect w-oxidized metabolites of 5-HETE and, hence, could not evaluate the influence of phenazine derivatives on this activity. While LTB4 is not the only chemotaxin present at infection sites, it is a significant endogenous chemotaxin when compared on a mole-for-mole basis with other substances (7). It has been demonstrated that LTB4 is the major PMNL chemotaxin produced by human alveolar macrophages (14). Possession by P. aeruginosa of a mechanism for preventing inactivation of LTB4 by human PMNL appears to be inconsistent with long-term survival of the organism, since increased chemotaxis of PMNL and macrophages in response to LTB4 should result in increased phagocytosis and bacterial killing at the infection site. However, it has been demonstrated that prior exposure of human PMNL to LTB4 leads to loss of their chemotactic responsiveness with respect to LTB4 (6). This loss has been attributed to down regulation of high-affinity LTB4 receptors on the cell surface (8, 26). As PMNL are the only cells known to inactivate LTB4 effectively by c-oxidation (13), the inability of PMNL exposed to pyocyanin to inactivate this compound may result in down regulation of PMNL LTB4 receptors in vivo and may reduce LTB4-mediated chemotaxis. Thus, an advantage may be conferred upon the pathogen for survival within the host. PMNL infiltration into infection sites in response to other chemotaxins could still occur, and these substances may then become more important than LTB4 as the principal chemotaxins. We conclude that pyocyanin appears to play an important role in altering the synthesis and subsequent metabolism of LTB4 by human PMNL. As LTB4 is considered to be a major mediator of inflammation, we conclude that bacterial pyocyanin may have a significant role as a virulence factor in this process. Thus, pyocyanin may contribute to the persistence of P. aeruginosa in chronic disease, particularly in lung infections such as cystic fibrosis and bronchiectasis.
2539
ACKNOWLEDGMENT This work was supported by Merck, Sharp and Dohme (Australia), Proprietary Limited. REFERENCES 1. Armstrong, A. V., D. E. S. Stewart-Tuli, and J. S. Roberts. 1971. Characterisation of the Pseudomonas aeruginosa factor that inhibits mouse-liver mitochondrial respiration. J. Med. Microbiol. 4:249-262. 2. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911917. 3. Bray, M. A. 1983. The pharmacology and pathophysiology of leukotriene B4. Br. Med. Bull. 39:249-254. 4. Brom, J., W. Konig, M. Stuining, M. Raulf, and M. Koller. 1987. Characterization of leukotriene B4-omega-hydroxylase activity within human polymorphonuclear granulocytes. Scand. J. Immunol. 25:283-294. 5. Davis, G., and P. J. Thornalley. 1983. Free radical production from the aerobic oxidation of reduced pyridine nucleotides catalysed by phenazine derivatives. Biochim. Biophys. Acta 724:456-464. 6. Goetzl, E. J., J. M. Boeynaems, J. A. Oates, and W. C. Hubbard. 1981. Stimulus-specificity of the chemotactic deactivation of human neutrophils by lipoxygenase products of arachidonic acid. Prostaglandins 22:279-288. 7. Goetzl, E. J., and W. C. Pickett. 1980. The human PMN leukocyte chemotactic activity of complex hydroxyeicosatetraenoic acids (HETEs). J. Immunol. 125:1789-1791. 8. Goldman, D. W., and E. J. Goetzl. 1984. Heterogeneity of human polymorphonuclear leukocyte receptors for leukotriene B4. J. Exp. Med. 159:1027-1041. 9. Gotoh, Y., H. Sumimoto, and S. Minakami. 1989. Purification and characterization of 20-hydroxy-leukotriene B4 dehydrogenase in human neutrophils. Eur. J. Biochem. 179:315-321. 10. Hassan, H. M., and I. Fridovich. 1980. Mechanism of the antibiotic action of pyocyanin. J. Bacteriol. 141:156-163. 11. Henson, P. M., J. E. Henson, C. Fittschen, G. Kimani, D. L. Bratton, and W. H. Riches. 1988. Phagocytic cells: degranulation and secretion, p. 363-390. In J. I. Gallin, I. M. Goldstein, and R. Snyderman (ed.), Inflammation: basic principles and clinical correlates. Raven Press, New York. 12. Knight, M., P. E. Hartman, Z. Hartman, and V. M. Young. 1979. A new method of preparation of pyocyanin and demonstration of an unusual bacterial sensitivity. Anal. Biochem. 95:19-23. 13. Lewis, R. A., and K. F. Austen. 1988. Leukotrienes, p. 121-128. In J. I. Gallin, I. M. Goldstein, and R. Snyderman (ed.), Inflammation: basic principles and clinical correlates. Raven Press, New York. 14. Martin, T. R., G. Raugi, T. L. Merritt, and W. R. Henderson. 1987. Relative contribution of leukotriene B4 to the neutrophil chemotactic activity produced by the resident human alveolar macrophage. J. Clin. Invest. 80:1114-1124. 15. Muller, P. K., K. Krohn, and P. F. Muhlradt. 1989. Effects of pyocyanine, a phenazine dye from Pseudomonas aeruginosa, on oxidative burst and bacterial killing in human neutrophils. Infect. Immun. 57:2591-2596. 16. Muller, M., and T. C. Sorrell. 1991. Production of leukotriene B4 and 5-hydroxyeicosatetraenoic acid by human neutrophils is inhibited by Pseudomonas aeruginosa phenazine derivatives. Infect. Immun. 59:3316-3318. 17. Muller, M., and T. C. Sorrell. 1991. Effect of pyocyanin and 1-hydroxphenazine on the assay of lactate dehydrogenase activity. J. Infect. Dis. 164:610-611. 18. Nugteren, D. H., and E. Hazelhof. 1973. Isolation and properties of intermediates in prostaglandin biosynthesis. Biochim. Biophys. Acta 326:448-461. 19. O'Flaherty, J. T. 1987. Phospholipid metabolism and stimulusresponse coupling. Biochem. Pharmacol. 36:407-412. 20. O'Flaherty, J. T., R. L. Wykle, J. Redman, M. Samuel, and M. Thomas. 1986. Metabolism of 5-hydroxyeicosatetraenoate by human neutrophils: production of a novel w-oxidized derivative.
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J. Immunol. 137:3277-3283. 21. Shak, S., and I. M. Goldstein. 1984. w-Oxidation is the major pathway for the catabolism of leukotriene B4 in human polymorphonuclear leukocytes. J. Biol. Chem. 259:10181-10187. 22. Shak, S., and I. M. Goldstein. 1985. Leukotriene B4 W-hydroxylase in human polymorphonuclear leukocytes: partial purification and identification as a cytochrome P-450. J. Clin. Invest. 76:1218-1228. 23. Soberman, R. J., J. P. Sutyak, R. T. Okita, D. F. Wendelborn, L. J. Roberts U, and K F. Austen. 1988. The identification and formation of 20-aldehyde leukotriene B4. J. Biol. Chem. 263: 7996-8002. 24. Stenson, W. F., and C. W. Parker. 1979. Metabolism of arachidonic acid in ionophore-stimulated neutrophils. J. Clin. Invest. 64:1457-1465. 25. Sumimoto, H., and S. Minakami. 1990. Oxidation of 20-hydrox-
INFECT. IMMUN.
yleukotriene B4 to 20-carboxyleukotriene B4 by human neutrophil microsomes. J. Biol. Chem. 265:4348-4353. 26. Vitkauskas, G., H. J. Showell, and E. L. Becker. 1980. Specific binding of synthetic chemotactic peptides to rabbit peritoneal neutrophils: effects on dissociability of bound peptide, receptor activity, and subsequent biologic responsiveness (deactivation). Mol. Immunol. 17:171-180. 27. Watson, D., J. MacDermot, R. Wilson, P. J. Cole, and G. W. Taylor. 1986. Purification and structural analysis of pyocyanin and 1-hydroxyphenazine. Eur. J. Biochem. 159:309-313. 28. Wilson, R., D. A. Sykes, D. Watson, A. Rutman, G. W. Taylor, and P. J. Cole. 1988. Measurement of Pseudomonas aeruginosa phenazine pigments in sputum and assessment of their contribution to sputum sol toxicity for respiratory epithelium. Infect. Immun. 56:2515-2517.
