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Killing of Mycobacterium tuberculosis by Neutrophils: A Nonoxidative Process Gerald S. Jones, Harold J. Amirault, and Burton R. Andersen
From the Departments of Medicine and Microbiology/Immunology, University of Illinois at Chicago, and ~ Medical Center, Chicago West Side VA
Mycobacterium tuberculosis is an intracellular pathogen that persists and replicates within nonactivated host macrophages. Activated macrophages can inhibit growth of mycobacteria [1-3]. Reactive oxygen metabolites have been implicated in aIactivated macrophage-mediated killing of M. tuberculosis although direct evidence is lacking [4]. Recent work, however, using a cell-free system consisting of xanthine oxidase and xanthine to produce superoxide anion and hydrogen peroxide has demonstrated that M. tuberculosis is highly resistant to killing by these species [5], although aIthough it is known that in sufficient quantities hydrogen peroxide (5 x 10-3 M) will tuberCIllosis [6]. Douvas et al. aI. [7] showed that human kill M. tuberctllosis monocyte-95 % viable by trypan blue exclusion, were adjusted to a final concentration of 1 X 10'7 neutrophils/ml and kept at 4°C until used.
Neutrophils were suspended at a concentration of 1 xX 10'7 cells/ml in HBSS containing 1% gelatin (Kodak, Rochester, NY) and 10% pooled human serum (HBSS-GS). The serum was from eight normal donors who were both purified protein derivative skin test-positive and -negative. The eight sera were pooled, aliquoted, and frozen at -80°C. Freshly drawn serum (from the neutrophil donor) was added to the pool as a source of complement, 1 part fresh to 4 parts frozen. The assays were performed in polystyrene tubes using 100 J.tl ~l of J.tl of neutrophils (l (1 X 107' the M. tuberculosis suspension, 500 ~l J.tl of HBSS-GS. When additional reagents were cells/ml), and 400 ~l used in various experiments the volume of the reaction mixture was held constant (1 ml) by decreasing the buffer volume. The ratio of neutrophils to M. tuberculosis was 10:1 unless otherwise stated. During the killing assay, the tubes were kept at 37°C on a rocker platform. Radiometric assay. The previously described radiometric assay for determining the viability of M. tuberculosis [6] used 100-J.tl 100-~l aliquots of the above-described reaction mixture removed aseptically at 0, 1, and 2 h during incubation at 37°C in a 5 % CO2-air mixture. These samples were diluted with 900 ~l J.tl of sterile water con(!2 % bovine albumin and 0.02 % Tween 80 and allowed to taining (f.2 stand for 15 min to lyse the neutrophils. Aliquots (100 J.tl) ~l) of the diluted samples were inoculated into radiolabeled Middlebrook 7H12 (Baetec 12A; Johnston Laboratories, Towson, MD) and inmedium (Bactec 14CCh2 was quantitated daily (Baccubated at 37°C, and the evolved 14C0 tec 460; Johnston Laboratories). The aspirated atmosphere was tee replaced with fresh sterile 5% 5 % CO2-air. The readings were expressed as growth index per 24 h. Growth index was read out directly on the instrument on a scale of 0-999 with a value of 100 correJ.tCi of 14C02 • These daily growth index readings sponding to 0.025 ~Ci C~ evolved and thus reflected the degree indicated the amount of CCh and rate of growth and respiration of M. tuberculosis. Differences in growth index values were proportional throughout the course of the reaction. Values were usually compared on about day 10 but before the point when values began to decrease due to depletion of the labeled substrate. Agar colony counts. Although previous studies have shown that there is a good correlation between the growth index of the radiometric assay and the number of viable mycobacteria [6], we performed parallel agar colony counts in many experiments to verify the observations. Colony-forming units were determined by plating 7HI0 agar and incubating these plates log dilutions ofthe samples on 7H10 5 % CO2-air for 21-28 days. The agar colony counts were at 37°C in 5% the original reacofthe expressed as colony-forming units per milliliter of tion mixture.
Phagocytosis M. tuberculosis (H37Rv) suspended in HBSS were incubated with isolated neutrophils at 37°C in a shaker bath at a neutrophil-tobacteria ratio of 1:10. Phagocytosis was measured by microscopically determining the percentage of neutrophils containing acid-faststaining bacilli.
Superoxide Assay Neutrophil superoxide production in response to particulate and soluble stimuli was determined using a superoxide assay based on the reduction of ferricytochrome C by superoxide (02-). (00. The specificity of the reduction was established by its inhibition by SOD. The assay was based on a 96-well microplate method described previously [13]. Briefly, ferricytochrome C reduction was determined by measuring the change in absorbance at 550 nm using an automatic microplate reader (Biotek 310; Biotek Instruments, Burling~l of a reaction mixture ton, VT). Wells were filled with 150 ILl ~l of the stimulus containing 4 mglml ferricytochrome C and 10 J.tl at its desired concentration, all in HBSS. A neutrophil suspension (50 J.tl; ~1; 5 X x 106 neutrophils/ml of HBSS) was added to the first 6 wells. An identical suspension of neutrophils containing 0.33 mglml SOD was added to the remaining wells. The reader was programmed to blank against the second set of wells for each stimulus. The absorbance at 550 nm was recorded as the difference in absorbance SOD-eontaining second set. between the first set of wells and the SOD-containing Immediately after adding the neutrophils the plate was read at 550 nm for the T = 0 values, where T = time. The plate was then incubated at 37°C on a microplate shaker (Dynatech Laboratories, Alexandria, VA) with gentle agitation and read on the microplate reader. The amount of 02- produced was expressed as nanomoles of 02- per 2.5 X 105 neutrophils per unit of time.
Results
Killing of M. tuberculosis in the presence of superoxide dismutase and catalase. The role of O2- and H20 2 in the neutrophil-mediated killing of M. tuberculosis was assessed by treating neutrophils with SOD and catalase. SOD enzymatically catalyzes the dismutation of O ~2- to H20 2 and O2, and H20 2 is reduced to O 2 and H 20 by catalase. Figure 1 shows that SOD, catalase, and both enzymes combined failed to diminish neutrophil-mediated killing of M. tuberculosis (H37Rv) as measured by the radiometric assay. Neutrophils lysed by sonication did not reduce the viability of M. tuberculosis (H37Rv) when compared with organisms tested alone. Killing ofM. tuberculosis in the presence of singlet oxygen and free radical inhibitors. Free radical and singlet oxygen inhibitors were added to a neutrophil-mediated killing assay to further examine the potential role of these oxygen intermediates in killing. Taurine Thurine is a scavenger for HOCI [14] and histidine for singlet oxygen [15]; deferoxamine mesylate prevents formation of 'OH by chelating free iron and inhibiting the Fenton reaction [16]. None of the inhibitors tested
Downloaded from http://jid.oxfordjournals.org/ at The University of British Colombia Library on June 25, 2015
Neutrophil-Mediated Killing Assays
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Killing of Mycobacterium tuberculosis by Neutrophils: A Nonoxidative Process Gerald S. Jones, Harold J. Amirault, and Burton R. Andersen
From the Departments of Medicine and Microbiology/Immunology, University of Illinois at Chicago, and ~ Medical Center, Chicago West Side VA
Mycobacterium tuberculosis is an intracellular pathogen that persists and replicates within nonactivated host macrophages. Activated macrophages can inhibit growth of mycobacteria [1-3]. Reactive oxygen metabolites have been implicated in aIactivated macrophage-mediated killing of M. tuberculosis although direct evidence is lacking [4]. Recent work, however, using a cell-free system consisting of xanthine oxidase and xanthine to produce superoxide anion and hydrogen peroxide has demonstrated that M. tuberculosis is highly resistant to killing by these species [5], although aIthough it is known that in sufficient quantities hydrogen peroxide (5 x 10-3 M) will tuberCIllosis [6]. Douvas et al. aI. [7] showed that human kill M. tuberctllosis monocyte-95 % viable by trypan blue exclusion, were adjusted to a final concentration of 1 X 10'7 neutrophils/ml and kept at 4°C until used.
Neutrophils were suspended at a concentration of 1 xX 10'7 cells/ml in HBSS containing 1% gelatin (Kodak, Rochester, NY) and 10% pooled human serum (HBSS-GS). The serum was from eight normal donors who were both purified protein derivative skin test-positive and -negative. The eight sera were pooled, aliquoted, and frozen at -80°C. Freshly drawn serum (from the neutrophil donor) was added to the pool as a source of complement, 1 part fresh to 4 parts frozen. The assays were performed in polystyrene tubes using 100 J.tl ~l of J.tl of neutrophils (l (1 X 107' the M. tuberculosis suspension, 500 ~l J.tl of HBSS-GS. When additional reagents were cells/ml), and 400 ~l used in various experiments the volume of the reaction mixture was held constant (1 ml) by decreasing the buffer volume. The ratio of neutrophils to M. tuberculosis was 10:1 unless otherwise stated. During the killing assay, the tubes were kept at 37°C on a rocker platform. Radiometric assay. The previously described radiometric assay for determining the viability of M. tuberculosis [6] used 100-J.tl 100-~l aliquots of the above-described reaction mixture removed aseptically at 0, 1, and 2 h during incubation at 37°C in a 5 % CO2-air mixture. These samples were diluted with 900 ~l J.tl of sterile water con(!2 % bovine albumin and 0.02 % Tween 80 and allowed to taining (f.2 stand for 15 min to lyse the neutrophils. Aliquots (100 J.tl) ~l) of the diluted samples were inoculated into radiolabeled Middlebrook 7H12 (Baetec 12A; Johnston Laboratories, Towson, MD) and inmedium (Bactec 14CCh2 was quantitated daily (Baccubated at 37°C, and the evolved 14C0 tec 460; Johnston Laboratories). The aspirated atmosphere was tee replaced with fresh sterile 5% 5 % CO2-air. The readings were expressed as growth index per 24 h. Growth index was read out directly on the instrument on a scale of 0-999 with a value of 100 correJ.tCi of 14C02 • These daily growth index readings sponding to 0.025 ~Ci C~ evolved and thus reflected the degree indicated the amount of CCh and rate of growth and respiration of M. tuberculosis. Differences in growth index values were proportional throughout the course of the reaction. Values were usually compared on about day 10 but before the point when values began to decrease due to depletion of the labeled substrate. Agar colony counts. Although previous studies have shown that there is a good correlation between the growth index of the radiometric assay and the number of viable mycobacteria [6], we performed parallel agar colony counts in many experiments to verify the observations. Colony-forming units were determined by plating 7HI0 agar and incubating these plates log dilutions ofthe samples on 7H10 5 % CO2-air for 21-28 days. The agar colony counts were at 37°C in 5% the original reacofthe expressed as colony-forming units per milliliter of tion mixture.
Phagocytosis M. tuberculosis (H37Rv) suspended in HBSS were incubated with isolated neutrophils at 37°C in a shaker bath at a neutrophil-tobacteria ratio of 1:10. Phagocytosis was measured by microscopically determining the percentage of neutrophils containing acid-faststaining bacilli.
Superoxide Assay Neutrophil superoxide production in response to particulate and soluble stimuli was determined using a superoxide assay based on the reduction of ferricytochrome C by superoxide (02-). (00. The specificity of the reduction was established by its inhibition by SOD. The assay was based on a 96-well microplate method described previously [13]. Briefly, ferricytochrome C reduction was determined by measuring the change in absorbance at 550 nm using an automatic microplate reader (Biotek 310; Biotek Instruments, Burling~l of a reaction mixture ton, VT). Wells were filled with 150 ILl ~l of the stimulus containing 4 mglml ferricytochrome C and 10 J.tl at its desired concentration, all in HBSS. A neutrophil suspension (50 J.tl; ~1; 5 X x 106 neutrophils/ml of HBSS) was added to the first 6 wells. An identical suspension of neutrophils containing 0.33 mglml SOD was added to the remaining wells. The reader was programmed to blank against the second set of wells for each stimulus. The absorbance at 550 nm was recorded as the difference in absorbance SOD-eontaining second set. between the first set of wells and the SOD-containing Immediately after adding the neutrophils the plate was read at 550 nm for the T = 0 values, where T = time. The plate was then incubated at 37°C on a microplate shaker (Dynatech Laboratories, Alexandria, VA) with gentle agitation and read on the microplate reader. The amount of 02- produced was expressed as nanomoles of 02- per 2.5 X 105 neutrophils per unit of time.
Results
Killing of M. tuberculosis in the presence of superoxide dismutase and catalase. The role of O2- and H20 2 in the neutrophil-mediated killing of M. tuberculosis was assessed by treating neutrophils with SOD and catalase. SOD enzymatically catalyzes the dismutation of O ~2- to H20 2 and O2, and H20 2 is reduced to O 2 and H 20 by catalase. Figure 1 shows that SOD, catalase, and both enzymes combined failed to diminish neutrophil-mediated killing of M. tuberculosis (H37Rv) as measured by the radiometric assay. Neutrophils lysed by sonication did not reduce the viability of M. tuberculosis (H37Rv) when compared with organisms tested alone. Killing ofM. tuberculosis in the presence of singlet oxygen and free radical inhibitors. Free radical and singlet oxygen inhibitors were added to a neutrophil-mediated killing assay to further examine the potential role of these oxygen intermediates in killing. Taurine Thurine is a scavenger for HOCI [14] and histidine for singlet oxygen [15]; deferoxamine mesylate prevents formation of 'OH by chelating free iron and inhibiting the Fenton reaction [16]. None of the inhibitors tested
Downloaded from http://jid.oxfordjournals.org/ at The University of British Colombia Library on June 25, 2015
Neutrophil-Mediated Killing Assays
701
700
700
600
600 500
500 )( I&J 1&1
c
~ ::J: :I:
~0~
II' GIl: Co:)
