Brief Com m unication
Biol Neonate 1992;62:63-66
Department of Pediatrics, Asahikawa Medical College, Asahikawa, Japan
An Increased Ability to Generate Hydrogen Peroxide in Certain Polymorphonuclear Leukocytes in Neonates
Key Words
Abstract
Polymorphonuclear leukocytes Oxidative burst Hydrogen peroxide
We performed a quantitive assay of the hydrogen peroxide generation in polymorphonuclear leukocytes using single cell analysis by flow cytometry. The study population included premature neonates (n = 10), term neonates (n = 28) and healthy adults (n = 29). The ability to generate hydrogen per oxide in neonatal polymorphonuclear leukocytes was totally reduced compared with adults, however, some polymorpho nuclear leukocytes in neonates demonstrated highly accen tuated hydrogen peroxide generation.
Introduction
Polymorphonuclear leukocytes (PML) have various functions in host defense against microbial infections [1]. Neonates have an increased incidence of bacterial infections when compared with older children and adults [2], Several abnormalities of neonatal neutrophil functions (chemotaxis, phagocyto sis, microbicidal activity and biochemistry) have been previously described. However, controversies exist in the significance of each defect between the reports; particularly oxida tive metabolism analyses have had conflicting
results [3-7], We therefore performed a quan titative assay of oxidative burst in PML ob tained from neonates using single cell analysis by flow cytometry. Subjects and Methods Study Population. Blood samples were obtained from 28 term neonates and 10 preterm neonates; they had no clinical abnormalities. In term neonates, the mean value for birth weight was 3,1 lOg (range 2,530— 3,720 g) and that for postnatal age was 6 days (range 4-8 days). In preterm neonates, the mean birth weight, postnatal age and gestational weeks were 1,880 g (range 1,570-2,400 g), 3 days (range 0-5 days) and 32
Daisuke U ida.M D Department of Pediatrics. Asahikawa Medical College Nishikagura 4-5-3-11 Asahikawa. Hokkaido 078 (Japan)
©1992 S. Karger AG. Basel 0006-3126/92/ 0621-006352.75/0
Downloaded by: Stockholm University Library 130.237.165.40 - 1/19/2019 8:37:35 AM
Daisuke Ueda Youko Sakata Kazuhiro Sasaki Hiroshi Azuma
64
Results
All subjects, from neonates to adults, pre sented heterogeneous H2C>2 generation in PML. Twenty-seven subjects of 29 healthy adults (93%) demonstrated a unimodal distri bution with a single peak: the majority of PML became fluorescence positive (fig. 1, solid line). The other 2 adults (7%) showed a bimodal PML distribution with a fluores cence-positive peak and a fluorescence-nega tive peak. In contrast, 17 subjects of 28 term neonates (60%) and 7 subjects of 11 preterm neonates (64%) presented a definite bimodal PML distribution. Namely, some PML be came fluorescence positive, and the remain ing showed low fluorescence intensity, or fluo rescence generation was negative (fig. 1, dot ted line). In addition, certain PML in neo nates demonstrated even higher fluorescence intensity than adult PML (fig. 1). The other 11 term neonates (40%) and 4 preterm neo nates (36%) demonstrated unimodal PML distribution. The mean (± SD) for each parameter of HyOy generation was obtained in each group (table 1). The F/T ratio in healthy adults was 0.909 ± 0.064; in term and in preterm neo nates they were 0.739 ± 0.138 and 0.755 ± 0.123, respectively (p < 0.01, compared with healthy adults). Peak fluorescence intensities in term neonates and preterm neonates ( 130.0 ± 22.0 and 130.4 ± 14.3. respectively) were greater than in healthy adults (101.2 ± 18.2, p < 0.01). Mean fluorescence intensities showed no significant differences between the three groups: 93.6 ± 11.2 in healthy adults, 95.6 ± 16.4 in term neonates and 93.3 ± 9.1 in preterm neonates. Cumulative fluores cence intensities in healthy adults, term and preterm neonates were 85.3 ± 12.9, 71.3 ± 22.2 and 71.2 ± 16.6, respectively (p < 0 .0 1 , compared with healthy adults). No significant difference was shown between term and pre-
U eda/Sakata/Sasakl/Azu ma
Hydrogen Peroxide Generation in Neonatal PML
Downloaded by: Stockholm University Library 130.237.165.40 - 1/19/2019 8:37:35 AM
weeks (range 28-36 weeks). Twenty-nine healthy adults were examined as normal controls. Complete blood count in each subject did not demonstrate eosinophilia. basophilia or abnormal leukocytes. Reagents. Phorbol myristate acetate (PMA) and Dulbecco’s phospahte-buffered saline (PBS) were ob tained from Sigma (St. Louis, Mo. USA and Gibco Grand Island. N.Y., USA), respectively. 2'.7'-Dichlorofluorcscin diacetate (DCFH-DA) and Immunolyse were purchased from Eastman Kodak (Rochester, N.Y. USA) and Coulter (Hialeah, Fla., USA), respec tively. PBS containing 5 mM glucose and 0.1 % gelatin was represented as PBSg and that containing 0.1% NaN 3 as PBSA. DCFH-DA and PMA were dissolved in ethanol at a concentration of 5 mM and 2 mg/ml. respectively. At the beginning of the assay. DCFH-DA and PMA were diluted with PBSg to 5 \iM and 25 gg/ml, respectively. A lysing solution, 0.87% NH4CI3 containing Immunolyse (2% vol/vol), was pre pared for erythrocytes elimination. Method. Oxidative burst of PML was examined by flow cytometric assay of DCFH oxidation which is quantitatively related to hydrogen peroxide (H 2O2) generation [8-10]. Heparinized whole blood (100 pi) was preincubated for 15 min with 5 p.V/ DCFH-DA in PBSg in a final volume of 2 ml by horizontal agitation in a shaking water bath at 37 ®C. PML were then stim ulated with PMA: 0.5 ml of EDTA (25 m.V/) and 10 pi of PMA (25 pg/ml) were added to the sample. After another incubation for 20 min. erythrocytes were elim inated with the lysing solution. Subsequently, PML were centrifuged (1.100 rpm 10 min, 4°C). washed with PBSg and resuspended with PBSA in a final vol ume of 2 ml. Flow microfluorometry was performed using CS20 (Shyowa Denko, Tokyo. Japan). A cytogram of the forward light scatter versus 90° light scatter and elec tronic gates were used to identify the PML population from lymphocytes and monocytes. The fluorescence intensity distribution of 10.000 PML was determined. Data are presented as the number of cells on the verti cal axis and the logarithm of green fluorescence inten sity on the horizontal axis. The channels including 99.5% of unstimulatcd PML (from 0 to n) were defined as fluorescence negative in each sample: the channels more than n were determined fluorescence positive. Four parameters of H20 2 generation were obtained: fluorescence-positive PML to total PML ra tio (F/T ratio), peak channel of fluorescence-positive PML, mean fluorescence intensity and cumulative flu orescence intensity, which was defined as: mean fluo rescence intensity X F/T ratio.
Fig. 1. Representative distribution patterns of fluo rescence intensity in PML stimulated by PMA. A A solid line indicates a unimodal distribution with a sin gle peak which is representative of adult PML: the majority of PML became fluorescence positive. B A dotted line indicates a definite bimodal distribution which is representative of neonatal PML: some PML arc highly fluorescence positive and the remaining show low fluorescence intensity or are fluorescence negative.
FLUORESCENCE
INTENSITY
Table 1. Various parameters of FLCF generation in PML in each population (means ± SD) Groups
Subjects (male:female)
F/T ratio
PFI
MFI
CFI
Adults Term neonates Preterm neonates
29(13:16) 28(17:11) 11 (7:4)
0.909 ±0.064 0.739 ±0.138* 0.755 ±0.123*
101.2 ± 18.2 .30.0 ±22.0* 130.4 ± 14.3*
93.6 ± 11.2 95.6± 16.4 93.9 ±9.1
85.3 ± 12.9 71.3 ± 22.2* 71.2 ± 16.6*
term neonates in all parameters. Each param eter in neonates presented no significant re gression to birth weight and to gestationl age.
Discussion
In our study, the mean value for the cumu lative fluorescence intensity in neonates was significantly lower than that in adults. And this lower index was mainly attributed to the lower percentage of fluorescence-positive PML compared with adults, because the mean value for the mean fluorescence inten sity showed no significant difference between neonates and adults. The data indicate that neonatal PML include a larger subpopulation which cannot generate LLCb by stimulation with PMA. However, the mean value for the
peak fluorescence intensity in neonates was significantly greater than that in adults. Namely, some neonatal PML became highly fluorescence positive, though neonatal PML had a large fluorescence-negative subpopula tion. It is suggested that certain neonatal PML have an increased ability to generate H2O2, which is even higher than adult PML. Thus neonatal PML is thought to be more heteroge neous in oxidative burst than adult PML [ 1 1 ]. We therefore concluded that the ability to generate H2O2 in neonatal PML was totally reduced as compared with adults, however, some PML in neonates demonstrated highlyaccentuated H2O2 generation. This character istic feature of neonatal PML may contribute to the various conflicting results in previous studies.
65
Downloaded by: Stockholm University Library 130.237.165.40 - 1/19/2019 8:37:35 AM
* p < 0.01. compared with adults. PFI = Peak fluorescence intensity; MFI = mean fluorescence intensity; CFI = cumulative fluorescence intensity.
1 Wilson CB: Immunologic basis for increased susceptibility of the neo nate to infection. J Pcdiatr 1986; 108:1-12. 2 Christensen RD. Rothstein G, Anstall HB. Bybee B: Granulocyte transfusion in neonates with bacte rial infection, neutropenia, and de pletion of mature neutrophils. Pedi atrics 1988:147:1-6. 3 Ambruso DR. Altenberg KM, John ston RB: Defective oxidative metab olism in newborn neutrophils: Dis crepancy between superoxide anion and hydroxy radical generation. Pe diatrics 1979:64:722-725. 4 Shigeoka AO. Charette PR, Wyman ML, Hill HR: Defective oxidative metabolic responses of neutrophils from stressed neonates. J Pediatr 1981:98:392-398.
66
5 Gahr M, Blanke R. Speer CP: Poly morphonuclear leukocyte function in term and preterm newborn in fants. Biol Neonate 1985:48:15-20. 6 Peden DB. Van Dyke K, Ardekani A. Mullett MD. Myerberg DZ, Van Dyke C: Diminished chemilumines cent responses of polymorphonu clear leukocytes in severely and moderately preterm neonates. J Pe diatr 1987;111:904-906. 7 Usmani SS, Schiessel JS. Sia CG, Kamran SK, Orncr SD: Polymor phonuclear leukocyte function in the preterm neonate: Effect of chronologic age. Pediatrics 1991:87: 675-679.
8 Bass DA. Parcc JW. Dechatelet LR. Szejada P, Seeds MC, Thomas M: Flow cytometric studies of oxidative product formation by neutrophils: A graded response to membrane stim ulation. J Immunol 1983:130:19101917. 9 Taga K. Seki H. Taniguchi N: Flow cytometric evaluation of neutrophil functions on microquantities of whole blood. Clin Immunol 1985; 17:490-498. 10 Hirabayashi Y. Taniuchi S, Kobayashi Y: A quantitive assay of oxida tive metabolism by neutrophils in whole blood using flow cytometry. J Immunol Methods 1985:82:253— 259. 11 Gallin JI: Human neutrophil hetero geneity exists, but is it meaningful? Blood 1984:63:977-983.
U cda/Sakata/Sasaki/Azu ma
Hydrogen Peroxide Generation in Neonatal PML
Downloaded by: Stockholm University Library 130.237.165.40 - 1/19/2019 8:37:35 AM
References
Biol Neonate 1992;62:63-66
Department of Pediatrics, Asahikawa Medical College, Asahikawa, Japan
An Increased Ability to Generate Hydrogen Peroxide in Certain Polymorphonuclear Leukocytes in Neonates
Key Words
Abstract
Polymorphonuclear leukocytes Oxidative burst Hydrogen peroxide
We performed a quantitive assay of the hydrogen peroxide generation in polymorphonuclear leukocytes using single cell analysis by flow cytometry. The study population included premature neonates (n = 10), term neonates (n = 28) and healthy adults (n = 29). The ability to generate hydrogen per oxide in neonatal polymorphonuclear leukocytes was totally reduced compared with adults, however, some polymorpho nuclear leukocytes in neonates demonstrated highly accen tuated hydrogen peroxide generation.
Introduction
Polymorphonuclear leukocytes (PML) have various functions in host defense against microbial infections [1]. Neonates have an increased incidence of bacterial infections when compared with older children and adults [2], Several abnormalities of neonatal neutrophil functions (chemotaxis, phagocyto sis, microbicidal activity and biochemistry) have been previously described. However, controversies exist in the significance of each defect between the reports; particularly oxida tive metabolism analyses have had conflicting
results [3-7], We therefore performed a quan titative assay of oxidative burst in PML ob tained from neonates using single cell analysis by flow cytometry. Subjects and Methods Study Population. Blood samples were obtained from 28 term neonates and 10 preterm neonates; they had no clinical abnormalities. In term neonates, the mean value for birth weight was 3,1 lOg (range 2,530— 3,720 g) and that for postnatal age was 6 days (range 4-8 days). In preterm neonates, the mean birth weight, postnatal age and gestational weeks were 1,880 g (range 1,570-2,400 g), 3 days (range 0-5 days) and 32
Daisuke U ida.M D Department of Pediatrics. Asahikawa Medical College Nishikagura 4-5-3-11 Asahikawa. Hokkaido 078 (Japan)
©1992 S. Karger AG. Basel 0006-3126/92/ 0621-006352.75/0
Downloaded by: Stockholm University Library 130.237.165.40 - 1/19/2019 8:37:35 AM
Daisuke Ueda Youko Sakata Kazuhiro Sasaki Hiroshi Azuma
64
Results
All subjects, from neonates to adults, pre sented heterogeneous H2C>2 generation in PML. Twenty-seven subjects of 29 healthy adults (93%) demonstrated a unimodal distri bution with a single peak: the majority of PML became fluorescence positive (fig. 1, solid line). The other 2 adults (7%) showed a bimodal PML distribution with a fluores cence-positive peak and a fluorescence-nega tive peak. In contrast, 17 subjects of 28 term neonates (60%) and 7 subjects of 11 preterm neonates (64%) presented a definite bimodal PML distribution. Namely, some PML be came fluorescence positive, and the remain ing showed low fluorescence intensity, or fluo rescence generation was negative (fig. 1, dot ted line). In addition, certain PML in neo nates demonstrated even higher fluorescence intensity than adult PML (fig. 1). The other 11 term neonates (40%) and 4 preterm neo nates (36%) demonstrated unimodal PML distribution. The mean (± SD) for each parameter of HyOy generation was obtained in each group (table 1). The F/T ratio in healthy adults was 0.909 ± 0.064; in term and in preterm neo nates they were 0.739 ± 0.138 and 0.755 ± 0.123, respectively (p < 0.01, compared with healthy adults). Peak fluorescence intensities in term neonates and preterm neonates ( 130.0 ± 22.0 and 130.4 ± 14.3. respectively) were greater than in healthy adults (101.2 ± 18.2, p < 0.01). Mean fluorescence intensities showed no significant differences between the three groups: 93.6 ± 11.2 in healthy adults, 95.6 ± 16.4 in term neonates and 93.3 ± 9.1 in preterm neonates. Cumulative fluores cence intensities in healthy adults, term and preterm neonates were 85.3 ± 12.9, 71.3 ± 22.2 and 71.2 ± 16.6, respectively (p < 0 .0 1 , compared with healthy adults). No significant difference was shown between term and pre-
U eda/Sakata/Sasakl/Azu ma
Hydrogen Peroxide Generation in Neonatal PML
Downloaded by: Stockholm University Library 130.237.165.40 - 1/19/2019 8:37:35 AM
weeks (range 28-36 weeks). Twenty-nine healthy adults were examined as normal controls. Complete blood count in each subject did not demonstrate eosinophilia. basophilia or abnormal leukocytes. Reagents. Phorbol myristate acetate (PMA) and Dulbecco’s phospahte-buffered saline (PBS) were ob tained from Sigma (St. Louis, Mo. USA and Gibco Grand Island. N.Y., USA), respectively. 2'.7'-Dichlorofluorcscin diacetate (DCFH-DA) and Immunolyse were purchased from Eastman Kodak (Rochester, N.Y. USA) and Coulter (Hialeah, Fla., USA), respec tively. PBS containing 5 mM glucose and 0.1 % gelatin was represented as PBSg and that containing 0.1% NaN 3 as PBSA. DCFH-DA and PMA were dissolved in ethanol at a concentration of 5 mM and 2 mg/ml. respectively. At the beginning of the assay. DCFH-DA and PMA were diluted with PBSg to 5 \iM and 25 gg/ml, respectively. A lysing solution, 0.87% NH4CI3 containing Immunolyse (2% vol/vol), was pre pared for erythrocytes elimination. Method. Oxidative burst of PML was examined by flow cytometric assay of DCFH oxidation which is quantitatively related to hydrogen peroxide (H 2O2) generation [8-10]. Heparinized whole blood (100 pi) was preincubated for 15 min with 5 p.V/ DCFH-DA in PBSg in a final volume of 2 ml by horizontal agitation in a shaking water bath at 37 ®C. PML were then stim ulated with PMA: 0.5 ml of EDTA (25 m.V/) and 10 pi of PMA (25 pg/ml) were added to the sample. After another incubation for 20 min. erythrocytes were elim inated with the lysing solution. Subsequently, PML were centrifuged (1.100 rpm 10 min, 4°C). washed with PBSg and resuspended with PBSA in a final vol ume of 2 ml. Flow microfluorometry was performed using CS20 (Shyowa Denko, Tokyo. Japan). A cytogram of the forward light scatter versus 90° light scatter and elec tronic gates were used to identify the PML population from lymphocytes and monocytes. The fluorescence intensity distribution of 10.000 PML was determined. Data are presented as the number of cells on the verti cal axis and the logarithm of green fluorescence inten sity on the horizontal axis. The channels including 99.5% of unstimulatcd PML (from 0 to n) were defined as fluorescence negative in each sample: the channels more than n were determined fluorescence positive. Four parameters of H20 2 generation were obtained: fluorescence-positive PML to total PML ra tio (F/T ratio), peak channel of fluorescence-positive PML, mean fluorescence intensity and cumulative flu orescence intensity, which was defined as: mean fluo rescence intensity X F/T ratio.
Fig. 1. Representative distribution patterns of fluo rescence intensity in PML stimulated by PMA. A A solid line indicates a unimodal distribution with a sin gle peak which is representative of adult PML: the majority of PML became fluorescence positive. B A dotted line indicates a definite bimodal distribution which is representative of neonatal PML: some PML arc highly fluorescence positive and the remaining show low fluorescence intensity or are fluorescence negative.
FLUORESCENCE
INTENSITY
Table 1. Various parameters of FLCF generation in PML in each population (means ± SD) Groups
Subjects (male:female)
F/T ratio
PFI
MFI
CFI
Adults Term neonates Preterm neonates
29(13:16) 28(17:11) 11 (7:4)
0.909 ±0.064 0.739 ±0.138* 0.755 ±0.123*
101.2 ± 18.2 .30.0 ±22.0* 130.4 ± 14.3*
93.6 ± 11.2 95.6± 16.4 93.9 ±9.1
85.3 ± 12.9 71.3 ± 22.2* 71.2 ± 16.6*
term neonates in all parameters. Each param eter in neonates presented no significant re gression to birth weight and to gestationl age.
Discussion
In our study, the mean value for the cumu lative fluorescence intensity in neonates was significantly lower than that in adults. And this lower index was mainly attributed to the lower percentage of fluorescence-positive PML compared with adults, because the mean value for the mean fluorescence inten sity showed no significant difference between neonates and adults. The data indicate that neonatal PML include a larger subpopulation which cannot generate LLCb by stimulation with PMA. However, the mean value for the
peak fluorescence intensity in neonates was significantly greater than that in adults. Namely, some neonatal PML became highly fluorescence positive, though neonatal PML had a large fluorescence-negative subpopula tion. It is suggested that certain neonatal PML have an increased ability to generate H2O2, which is even higher than adult PML. Thus neonatal PML is thought to be more heteroge neous in oxidative burst than adult PML [ 1 1 ]. We therefore concluded that the ability to generate H2O2 in neonatal PML was totally reduced as compared with adults, however, some PML in neonates demonstrated highlyaccentuated H2O2 generation. This character istic feature of neonatal PML may contribute to the various conflicting results in previous studies.
65
Downloaded by: Stockholm University Library 130.237.165.40 - 1/19/2019 8:37:35 AM
* p < 0.01. compared with adults. PFI = Peak fluorescence intensity; MFI = mean fluorescence intensity; CFI = cumulative fluorescence intensity.
1 Wilson CB: Immunologic basis for increased susceptibility of the neo nate to infection. J Pcdiatr 1986; 108:1-12. 2 Christensen RD. Rothstein G, Anstall HB. Bybee B: Granulocyte transfusion in neonates with bacte rial infection, neutropenia, and de pletion of mature neutrophils. Pedi atrics 1988:147:1-6. 3 Ambruso DR. Altenberg KM, John ston RB: Defective oxidative metab olism in newborn neutrophils: Dis crepancy between superoxide anion and hydroxy radical generation. Pe diatrics 1979:64:722-725. 4 Shigeoka AO. Charette PR, Wyman ML, Hill HR: Defective oxidative metabolic responses of neutrophils from stressed neonates. J Pediatr 1981:98:392-398.
66
5 Gahr M, Blanke R. Speer CP: Poly morphonuclear leukocyte function in term and preterm newborn in fants. Biol Neonate 1985:48:15-20. 6 Peden DB. Van Dyke K, Ardekani A. Mullett MD. Myerberg DZ, Van Dyke C: Diminished chemilumines cent responses of polymorphonu clear leukocytes in severely and moderately preterm neonates. J Pe diatr 1987;111:904-906. 7 Usmani SS, Schiessel JS. Sia CG, Kamran SK, Orncr SD: Polymor phonuclear leukocyte function in the preterm neonate: Effect of chronologic age. Pediatrics 1991:87: 675-679.
8 Bass DA. Parcc JW. Dechatelet LR. Szejada P, Seeds MC, Thomas M: Flow cytometric studies of oxidative product formation by neutrophils: A graded response to membrane stim ulation. J Immunol 1983:130:19101917. 9 Taga K. Seki H. Taniguchi N: Flow cytometric evaluation of neutrophil functions on microquantities of whole blood. Clin Immunol 1985; 17:490-498. 10 Hirabayashi Y. Taniuchi S, Kobayashi Y: A quantitive assay of oxida tive metabolism by neutrophils in whole blood using flow cytometry. J Immunol Methods 1985:82:253— 259. 11 Gallin JI: Human neutrophil hetero geneity exists, but is it meaningful? Blood 1984:63:977-983.
U cda/Sakata/Sasaki/Azu ma
Hydrogen Peroxide Generation in Neonatal PML
Downloaded by: Stockholm University Library 130.237.165.40 - 1/19/2019 8:37:35 AM
References
