INFECTION AND IMMUNITY, May 1979, p. 399-403
Vol. 24, No. 2
Inhibitory Effect of Pseudomonas aeruginosa on the Phagocytic and Killing Activity of Rabbit Polymorphonuclear Leukocytes: Mechanisms of Action of a Polymorphonuclear Leukocyte Inhibitor SHIGEO NONOYAMA,' HITOSHI KOJO,' YASUHIRO MINE,' MINORU NISHIDA,' SACHIKO GOTO,2 AND SHOGO KUWAHARA2 Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan,' and Department of Microbiology, Toho University School ofMedicine, Tokyo, Japan2 Received for publication 18 February 1979
The polymorphonuclear leukocyte (PMN) inhibitor isolated from a strain of Pseudomonas aeruginosa which is resistant to the phagocytic and killing activities of rabbit PMN inhibited migration of PMN and engulfment of latex particles by PMN. In studies of the bactericidal metabolism of PMN, the PMN inhibitor did not inhibit the intracellular activity and extracellular release of lysosomal enzymes. However, the PMN inhibitor caused a decrease of Nitro Blue Tetrazolium reduction. The PMN inhibitor had a cytotoxic effect on PMN and inhibited ["4C]tyrosine uptake in intact PMN, but did not inhibit ["4C]uridine and [14C]thymine uptake. The PMN inhibitor had no inhibitory effect on protein synthesis in cell extracts. Pseudomonas aeruginosa produces a number of toxic substances. Since the discovery of exotoxin A was originally described by Liu et al. (79), various workers have demonstrated the importance of this toxin in the pathogenicity of P. aeruginosa. In our preceding paper, the relationship between P. aeruginosa and polymorphonuclear leukocytes (PMN), which play an important role in host defense mechanisms, is reported (11). We find that virulent strains of P. aeruginosa produce an extracellular substance of high molecular weight that inhibits the phagocytic and killing activities of PMN. This substance is purified from culture filtrate by precipitation with (NH4)2SO4 and successive chromatography on phosphocellulose, diethylaminoethyl-cellulose, and Sephadex G-100. A homogenous preparation is obtained as judged by disc gel electrophoresis. The substance has the properties of a protein with a molecular weight of 65,000 and is inactivated by heat and by proteolytic enzyme treatments. This paper describes studies of the mechanisms of the inhibitory action of an extracellular substance (the PMN inhibitor) against the phagocytic and killing activities of PMN.
The inhibitor (20 ml) was purified from culture filtrates (3 liters) of PMN-resistant Pseudomonas strain 7005 by precipitation with (NH)2SO4 followed by dialysis and chromatography on phosphocellulose, diethylaminoethyl-cellulose, and Sephadex G-100. This purified PMN inhibitor showed an absorbance at 280 nm of 0.128 (this concentration is refered to as 128 A2w/ml) and inhibited the phagocytic and killing activities of PMN. In control experiments where the PMN inhibitor was omitted, E. coli strain 303 was killed by PMN and the viable cell counts decreased from 3 x 106 to 2 x 103 colony-forming units per ml after 4 h of incubation. In the presence of the PMN inhibitor, there was no fall in the count of E. coli strain 303 (3 x 106 colony-forming units per ml). Isolation of PMN. PMN were isolated from pentoneal exudates of rabbits injected with a 0.1% glycogen solution as described previously (11, 12). Migration inhibition test. PMN migration was tested by the methods described by Clausen (5) and Aster et al. (2) with some modifications. A 2% agarose solution was prepared with distilled water and autoclaved. When the solution was cooled to 450C, an equal volume of Hanks balanced salt solution (HBSS) was added. Rabbit serum was then added to give a
final serum concentration of 10%. A 10-ml amount of this mixture was poured into disposable plastic petri dishes. Four wells were made in the medium, 8 mm in diameter. A 0.2-ml quantity of PMN suspension (5 x 108 cells/ml) was placed in all wells and incubated at 370C for 32 h in an atmosphere of 5% C02. The PMN inhibitor was added to the well and agarose medium MATERIALS AND METHODS to test for inhibition of PMN migration. Engulfment of latex by PMN. A PMN suspension PMN inhibitor. The PMN inhibitor was obtained by the method described by Nonoyama et al. (12a). containing 8 x 106 cells per ml of HBSS was shaken 399
NONOYAMA ET AL.
with latex (107 particles per ml) at 370C for 30 min. A portion of the mixture was taken, smeared on a glass slide, and stained with Giemsa stain. The number of PMN engulfing latex and the average number of latex engulfed by PMN were counted under a microscope. Measurement of lysosomal enzyme activities. fl-Glucuronidase and fi-galactosidase activities were measured by the methods of Stahl (15) and Meislar (10), respectively. A 5-ml amount of PMN suspension (107 cells per ml) in HBSS was incubated for 4 h at 370C with or without 0.5 ml of the PMN inhibitor (128 Amss/ml). PMN were collected by centrifugation, resuspended in 1 ml of HBSS, and sonicated at 15 kW for 3 min. PMN sonicates were assayed forfl-glucuronidase and /1-galactosidase activities. One unit of ,Bgalactosidase is defined as the amount of enzyme which catalyzes the hydrolysis of 1 ILM of p-nitrophenyl-,8-D-galactoside per min, whereas one unit of /?-glucuronidase is defined as the activity which catalyzes the release of 1 jumol of phenolphthalein per h from phenolphthalein glucuronide. NBT reduction test. Nitro Blue Tetrazolium (NBT) reduction was measured by the method of Baehner et al. (3). The assay mixture contained 1 ILM of potassium cyanide, 4 mg of NBT, 2 x 106 particles of latex spherules (when indicated), and 107 cells of PMN or sonicated PMN with or without 0.4 ml of the PMN inhibitor (128 An0/ml) in 1 ml of HBSS. After 1 h of incubation at 370C, reaction was stopped by the addition of 10 ml of 0.5 N hydrochloric acid, and the mixture was then centrifuged at 1,000 x g for 15 min. The resultant precipitate was extracted for 10 min with 2 ml of pyridine in a boiling-water bath. The optical density of the reduced NBT was determined at A51,5. Uptake of amino acid by PMN. To 1 ml of PMN suspension in HBSS containing 2 x 106 cells per ml, 10 ,ul of ['4C]tyrosine (100 ,uCi/ml; specific activity, 100 mCi/mmol), [14C]uridine (100 ,Ci/ml; specific activity, 51 mCi/mmol), or ['4C]thymine (100 ,Ci/ml; specific activity, 51 mCi/mmol) was added. PMN incorporation of radioactivity was measured in the scintillation counter (Packard, Tricarb, model 3,380) by the procedures of Umeda et al. (16). Protein synthesis in the cell extract. PMN (3 x 107 cells) were lysed by adding 1 ml of sterile distilled water, and the lysate was centrifuged at 3,000 rpm for 10 min (1, 6). A 1-ml amount of the supernatant was added to 20 1d of ['4C]tyrosine (100 ,uCi/ml; specific activity, 100 mCi/mmol) and incubated for the appropriate time at 260C. Proteins were then precipitated by adding 5% trichloroacetic acid and hydrolyzed by heating at 1000C for 15 min. After chilling at 00C for 30 min, the protein precipitates were filtered through 0.45-am membranes (Millipore Corp.) and assayed for radioactivity.
RESULTS Effect on PMN migration. The effect of the PMN inhibitor against PMN migration is shown in Fig. 1. When PMN inhibitor was added to agarose medium, the migration of PMN was strongly inhibited as compared with that of the
FIG. 1. Inhibition of rabbit PMN migration by PMN inhibitor in agarose medium. A 1-ml amount of the 128-A2so/ml PMN inhibitor or the 32-A2w/ml PMN inhibitor was added to 9 ml of agarose medium, respectively. The PMN suspension (5 x 108 cells per ml) of 0.2 ml was placed in the wells. The migration area was measured.
control without the PMN inhibitor. A dose-response relationship was shown as the concentration increased. Similarly, the PMN migration was inhibited when the PMN inhibitor was added to the PMN suspension in the well. The migration inhibition could not be reversed by adding ATP (1 mM) to the agarose medium and
PMN suspension. Effect on engulfment of latex by PMN. The effect of the PMN inhibitor on the engulfment of latex by PMN is shown in Fig. 2. The PMN inhibitor (43 A2so/ml) inhibited engulfment of latex by PMN. The ratio of PMN engulfing latex to the total numbers of PMN counted under a microscope was decreased by the PMN inhibitor, i.e., from 57 to 37% (after 10 min of incubation), 53 to 40% (20 min), 58 to 40% (30 min), and 62 to 30% (60 min) (Fig. 2A). The PMN inhibitor did not affect the average number of latex engulfed per PMN (Fig. 2B). Effect on bactericidal activity of sonicated PMN. Figure 3 shows the inhibitory effect of the PMN inhibitor on bactericidal activity of sonicated PMN. The PMN suspension in HBSS (108 cells per ml) was sonicated for 2 min and centrifuged at 3,500 rpm for 10 min. The influence of the PMN inhibitor on the bactericidal activity of the supernatant fluid against Escherichia coli strain 303 was evaluated. When the PMN inhibitor was omitted from the test medium, E. coli strain 303 was killed by the supernatant of sonicated PMN, and the viable cell counts decreased from 6 x 106 to 5 x 105 colony-forming units per ml after a 180-mnu
VOL. 24, 1979
MECHANISMS OF ACTION OF PMN INHIBITOR
X w E-4
60 10 20 INCUBATION TIME (MIN)
FIG. 2. Effect of PMN inhibitor on PMN engulfing latex and on number of latex engulfed by PMN. The PMN inhibitor (128 Aws/ml) of ml was added to 2 ml of PMN suspension (8 x 10t cells per ml) and was shaken with latex (107 particles per ml). The 2()0 cells were observed under a microscope and the number of PMN engulfing latex (A) and average number of latex particles engulfed by PMN (B) were calculated.
gated by studying its effects on lysosomal enzymes and in the NBT reduction test. Table 1 shows the effect of the PMN inhibitor against the intracellular activities of fl-glucuronidase and /3-galactosidase, and Fig. 4 shows the effect of the PMN inhibitor against extracellular release of these enzymes. The PMN inhibitor inhibited neither the intracellular activity nor extracellular release of these enzymes. zu PMN-INHIBI The effect of the PMN inhibitor (51 A2ws/ml) in¢ 06 against NBT reduction on intact PMN and son\ icated PMN is shown in Table 2. NBT reduction was compared in intact PMN both with and Ed4 without latex. The NBT reduction of PMN was decreased to 88% (with latex) and 79% (without latex) by adding the PMN inhibitor. However, 60 180 no inhibitory effect of the PMN inhibitor was INCUBATION TIME (MIN) seen in the NBT reduction test with sonicated FIG. 3. Effect of PMN inhibitor on the bactericidal PMN. activity of sonicated PMN against E. coli strain 303. Cytotoxic effect on PMN. The cytotoxicity The PMN inhibitor (128 Ameo/ml, 1 ml) was added to 4 ml of the supernatant fluid ofsonicated PMN which of the PMN inhibitor to PMN was investigated by trypan blue staining (Fig. 5). A cytotoxic was equivalent to 3 x 101 cells per ml of the PMN suspension. The bactericidal activity of the superna- effect was observed 15 min after treatment with WITHOUT
tant fluid against E. coli strain 303 was measured as decrease in colony-forming units.
TABLE 1. Effect of the PMN inhibitor on intracellular lysosomal enzymes of rabbit PMN
incubation. In the presence of the PMN inhibitor Lysosomal enzyme (26 Ameo/ml), the viable cells of E. coli strain 303 PMN inhibitor f-Glucronidid not decrease (6 x 106 colony-forming units fl-Galactosidase dase per ml), indicating that the PMN inhibitor inUntreated PMN 6.8a (100) b 5.3 x 10-4a (100) b terfered with the bactericidal activity of the 6.4 (96) 5.9 x 10-4 (111) Treated PMN PMN lysate. Effect on several antimicrobial systems. Enzyme activity (units). b The inhibitory effect of the PMN inhibitor Ratio of enzyme activity of treated PMN to unagainst PMN bactericidal activity was investi- treated PMN (100).
NONOYAMA ET AL. A)
PMH-INHIBITORE 1' 4
| -@ WITH
2 1 INCUBATION TIME (HOUR)
FIG. 4. Effect of the PMN inhibitor on the secretion of lysosomal enzymes from rabbit PMN. PMN were incubated with or without PMN inhibitor in the same conditions as described in Table 1. Portions (1 ml) were withdrawn at the indicated time and centrifuged at 1,000 x g for 15 min. A 0.1-ml amount of the resultant supernatant was used in enzyme assays.
Inhibition of ['4C]tyrosine uptake by PMN was first noted at 20 min after treatment with the PMN inhibitor (26 Am.s/ml). However, ["C]tyrosine incorporation into trichloroacetic acidprecipitable material of the PMN extract was not diminished in the presence of the PMN inhibitor (26 A2mo/ml), indicating that the PMN inhibitor did not inhibit protein synthesis. Moreover, ["4C]uridine and ['4C]thymine uptake by PMN was not inhibited by the PMN inhibitor (26 A2so/ml) (Fig. 7).
DISCUSSION Liu (7-9) and Pavlovskis et al. (4, 13) have demonstrated that exotoxin A (PA toxin) produced by P. aeruginosa was inactivated by heating and proteolytic enzyme. Crude fractions of PA toxin preparations are shown to inhibit the TABLE 2. Effect of the PMN inhibitor on NBT killing activity of PMN, but not the uptake of reduction bacteria ingested by PMN. Relatively pure PA Intact PMN So)ted toxin has no effect on ingestion or killing of Treatment With latex Without la- PMN(lathbacteria by PMN (13). Pavlovskis et al. (14) have reported that PA toxin inhibits ['4C]uridine uptake in Vero cell culture and protein synthesis Control 0.642a (100)b 0.616 (100) 0.049 (100) PMN 0.568 (88) 0.487 (79) 0.050 (100) in mouse liver in vivo. Furthermore, Iglewski and Kabat (6) described PA toxin that inhibits inhibitor cell-free protein synthesis and exerts an enzya A515 of the reduced NBT. b Ratio of optical density of the treatment by the matic activity which appears to be similar to the nicotinamide adenine dinucleotide-dependent PMN inhibitor to control (100). adenosine diphosphate-ribosyl transferase activity. 100 In our previous study, it was reported that PMN inhibitor isolated from P. aerugipurified t 80 nosa resistant to phagocytic and killing activities 0