Nonopsonic Phagocytosis of Strains of Pseudomonas Aeruginosa from Cystic Fibrosis Patients DAVID P
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INFECTION AND IMMUNITY, Mar. 1984, p. 1006-1011 Vol. 43, No. 3 0019-9567/84/031006-06$02.00O0 Copyright © 1984, American Society for Microbiology Nonopsonic Phagocytosis of Strains of Pseudomonas aeruginosa from Cystic Fibrosis Patients DAVID P. SPEERT,12* FERESHTEH EFTEKHAR,1 AND MARTIN L. PUTERMAN"3 Division of Infectious Diseases, Department of Pediatrics,' Department of Microbiology,2 and Faculty of Commerce,3 University of British Columbia, Vancouver, British Columbia, Canada V6H 3V4 Received 15 August 1983/Accepted 14 October 1983 Pseudomonas aeruginosa is the predominant respiratory pathogen in patients with cystic fibrosis, but its Mechanism of persisting in pulmonary secretions is poorly understood. We observed that three nonmucoid cystic fibrosis P. aeruginosa strains were phagocytized and one strain resisted phagocytosis by human polymorphonuclear leukocytes in the absence of serum. Phagocytosis was assessed by luminol-enhanced chemiluminescence, inspection of stained smears, bactericidal assay, reduction of nitroblue tetrazolium dye, and electron microscopy. Phagocytosis, determined by visual inspection, occurred at 35°C but not at 4°C. Nonopsonic phagocytosis was inhibited most efficiently by D-mannose, mannose-containing saccha- rides, and D-fructose. Opsonin-dependent phagocytosis of P. aeruginosa and of zymosan was not markedly inhibited by mannose, suggesting different leukocyte receptors for nonopsonic and opsonic phagocytosis. Pseudomonas aeruginosa has lately emerged as an impor- serum was decanted after centrifugation, pooled, and frozen tant pathogen in individuals with impairment of host de- in aliquots at -70°C for use in separate experiments. fenses due to cystic fibrosis, thermal injury, and malignant PMN. Heparinized venous human blood was layered on tumors (21, 25). Other investigators have defined the opsonic mono-poly resolving medium (Flow Laboratories, Inc., requirements for phagocytosis and killing of P. aeruginosa McLean, Va.) and centrifuged at 300 x g for 30 min. The by human polymorphonuclear leukocytes (PMN) (2, 16, 28) PMN layer was carefully pipetted off, washed twice in and by macrophages (20) and have attempted to explain why GHBSS, and adjusted by hemocytometer to contain 106 strains from patients with cystic fibrosis resist phagocytosis PMN per ml. Approximately 96% of the cells obtained were (1, 23). In studies with cystic fibrosis P. aeruginosa isolates, PMN. Greater than 98% were viable as assessed by trypan we observed that phagocytosis occurred in the absence of blue dye exclusion. serum and was inhibited by certain sugars. Observations on Sugars. D-Mannose, oa-methyl-D-mannoside (aMM), man- the nonopsonic phagocytosis of P. aeruginosa by human nan, cx-methyl-D-galactoside, D-glucose, and D-fructose PMN form the basis of this report. were all obtained from Sigma Chemical Co. (St. Louis, Mo.). Solutions (10%) were prepared in GHBSS, filter sterilized, MATERIALS AND METHODS kept at 4°C, and diluted before each experiment. Bacterial strains. Mucoid strains of P. aeruginosa were Zymosan preparation. The method of Mills et al. (13) was cultured from the sputum of patients with cystic fibrosis and followed for zymosan preparation. Zymosan (Sigma) was identified by oxidase reaction, growth at 42°C, pigment boiled for 1 h in HBSS (10 mg/ml), centrifuged, cooled, production, and the API 20E system (Analytab Products, resuspended in phosphate-buffered saline, and opsonized (1 Inc., Plainville, N.Y.). They were serotyped by using the part in 3 parts 100% pooled human serum) for 15 min. It was Difco typing system (Difco Laboratories, Detroit, Mich.). again centrifuged and resuspended in HBSS. Strain P-1 (type 9/10) was from the University of Minnesota Luminol-enhanced CL. For luminol-enhanced chemilumi- Hospitals, Minneapolis. Strains C-1 (type 6/9/10), C-46 (type nescence (CL) a modification of the method of Mills et al. 9/10), and C-96 (nontypable) were from British Columbia's (13) was used. Glass scintillation vials were dark adapted Children's Hospital, Vancouver. Nonmucoid spontaneous and then filled with 4.4 ml of GHBSS; to some, one or laboratory revertants were frozen at -70°C in Mueller- another of the above sugars was added. To this was added Hinton broth with 8% dimethyl sulfoxide and used as seeds 10-7 M luminol (5-amino-2,3 dihydro-1,4-phthalazinedione for each experiment. For all experiments, nonmucoid rever- [Sigma]) dissolved in dimethyl sulfoxide and further diluted tant bacteria were grown on Mueller-Hinton agar at 35°C for in phosphate-buffered saline and 0.1 ml of a suspension of 18 h, removed with a sterile swab, washed thrice in phos- 109 bacteria per ml. The vials were further dark adapted. phate-buffered saline (pH 7.4), and adjusted spectrophoto- Under red light illumination, 1 ml of 105 PMN per ml was metrically (Spectronic 20; Bausch & Lomb, Inc., Rochester, added to each vial, which was then agitated manually for 20 N.Y.) to 109 bacteria per ml in Hanks balanced salt solution s. Immediately thereafter, CL was measured at ambient (HBSS) with 0.1% gelatin (GHBSS). All four strains were temperature for 20 s in a Beckman LS-6800 liquid scintilla- very sensitive to the bactericidal effect of pooled normal tion counter in which the photomultiplier tubes had been human serum; >99% killing occurred after ihcubation at adjusted out of coincidence. CL was measured every 12 min 35°C for 60 min in 5% serum. in each of 22 vials, and the cycle was repeated 10 times. All Serum. Venous blood was drawn from five healthy adults samples were analyzed in duplicate, and the mean CL value and allowed to clot for 30 min at room temperature. The was computed. In some experiments 10-6 M luminol and 5 mg of opsonized zymosan were added to the vials. All experiments were repeated on at least 3 separate days with * Corresponding author. PMN from different donors. Peak CL varied substantially 1006 VOL. 43, 1984 NONOPSONIC PHAGOCYTOSIS OF P. AERUGINOSA 1007 from day to day, but the differences among experimental TABLE 1. Influence of various saccharides on phagocytosis of conditions were reproducible. Results of typical experiments nonopsonizied P. aeruginosa strain P-1 are reported. Saccharidea Avg bacteria/ Avg square root Bactericidal assay. For the bactericidal assay, a modifica- PMNb of bacteria/PMN' tion of the method of Quie et al. (18) was used in which Control (no saccharide) 14.08 3.64 serum was omitted from the phagocytosis mixture. To a-Methyl-D-galactoside 13.21 3.51 polypropylene snap-cap tubes (12 by 75 mm; BD Labware, D-Fucose 12.41 3.44 Oxnard, Calif.) were added 106 PMN and 106 bacteria with D-Glucose 10.59 3.08 or without one of several sugars. Tubes were rotated end D-Mannose 9.04 2.82 over end at 35°C, and samples were removed at 0, 30, and aMM 7.98 2.62 120 min and diluted in ice-cold water to lyse PMN. Serial D-Fructose 7.66 2.63 dilutions were made and viable bacteria were counted by the Mannan 1.10 0.76 spread-plate technique. In some tubes GHBSS was substi- a All saccharides were at 1% (wt/vol). tuted for PMN and some were held stationary. Using this b Averages are from replications of the experiment on 2 separate method with normal PMN, there is consistently >90% killing days. of Staphylococcus aureus 502A and serum-resistant P. aeru- c The standard error for tests of pairwise difference is 0.13. ginosa strain PAO1 in 30 min in the presence of 8% serum Pairwise differences of the square root exceeding 0.40 are significant and no killing in the absence of serum (D. P. Speert, Y. Kim, at the 0.05 level. and R. Grandy, Program Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 21st, Chicago, Ill., abstr. no 349, 1981). Visual assessment of phagocytosis. To polypropylene tubes Samples were viewed with a Phillips 400 electron micro- at 80 kV. 75 mm; BD Labware) were added 107 bacteria and 105 scope operating (12 by Statistics. Data in Tables 1 to 4 were analyzed by analysis PMN with or without one of several sugars. The tubes were of variance. The variable was taken to be the end over end for 60 min at at in one set dependent rotated 35°C (or 4°C root the of bacteria/PMN. This transforma- and then at 26 x g for 10 min. square of count of experiments) centrifuged was so that statistical inference based on the were twice in 0.5 tion used The cells gently washed more, resuspended normal distribution would be valid (3). Differences were ml of GHBSS, and deposited on a glass slide, using a Products considered significant if the simultaneous P-value based on Cytospin 2 cytocentrifuge (Shandon Southern for all tests in a experi- at 600 rpm for 5 min. The slides Bonferroni's method (3) particular Ltd., Astmoor, England) ment was <0.05. Computations were performed with BMDP were air dried, heat fixed, and stained with crystal violet. and P2V. The identity of each slide was unknown to the observer at computer programs P1D, P7D, the time of the bacterial count, and the number of bacteria RESULTS within the cytoplasm of each of 50 cells was quantitated. Although absolute counts varied from day to day, relation- P. aeruginosa strain P-1 was well phagocytized by human ships among experimental conditions were reproducible. PMN in the absence of serum as assessed by visual inspec- NBT reduction. A 0.2-ml portion of a PMN suspension tion (Table 1) and by induction of CL (Fig. 1). Phagocytosis (106/ml) was placed on a clean glass cover slip, incubated at was significantly inhibited (P < 0.0018) when mannan, D- 35°C for 30 min, and rinsed with phosphate-buffered saline.