Clin. exp. Immunol. (1990) 81, 156-159
An increase in polymorphonuclear leucocyte chemotaxis accompanied by a change in the membrane fluidity with age during childhood K. YASUI, M. MASUDA*, T. TSUNO, T. MATSUOKA, A. KOMIYAMA, T. AKABANE & K. MURATA* Department of Paediatrics, Shinshu University School of Medicine, Matsumoto, and * Department of Clinicolaboratory Medicine, Kansai Medical University, Osaka, Japan (Acceptedfor publication I February 1990)
SUMMARY Leucocyte membrane fluidity affects cell deformability as well as the accessibility of receptors and the degree of their exposure on the membrane. These effects modulate the subsequent cellular responsiveness. We have assayed membrane fluidity of polymorphonuclear leucocytes of children, using an excimer-forming lipid technique in flow cytometry, and evaluated its relation to their chemotaxis to a formyl peptide. We report that membrane fluidity and chemotaxis of polymorphonuclear leucocytes are increased with age. These findings may have important implications for the physiological processes in polymorphonuclear leucocyte motility during childhood. Keywords N-formylmethionyl-leucyl-phenylalanine leucocytes
The impaired chemotaxis of polymorphonuclear leucocytes (PMNL) in human newborns and infants is an important cause of increased morbidity and mortality due to bacterial infections (Klein et al., 1977; Laurenti et al., 1980). The mechanisms have been studied, but little is understood of their operation so far (Wilson, 1986; Hill, 1987). Miller (1978) has indicated that neonatal PMNL fail to deform normally following chemotactic factor stimulation. Erythrocytes from human neonates also differ from those of adults in having decreased filterability (Linderkamp, Hammer & Miller, 1986). Crespo, Bifano & Freedman (1988) found that erythrocytes from human neonates have decreased membrane lipid fluidity, which is related to increased cell rigidity and impaired deformability. Therefore, decreased filterability of neonatal PMNL may be explained at least in part by immaturity of the PMNL membrane. Repo et al. (1980) indicated that the impaired chemotaxis of neonatal PMNL was due not only to defective deformability but also to a defect in cellular responsiveness. The initiation of some PMNL responses, for example chemotaxis, is conducted via specific receptors on the membrane (Williams et al., 1977; Yasui et al., 1987). The membrane functional proteins, which include receptors, are also regulated by membrane fluidity (Galla & Hartmann, 1980). Manipulation of PMNL membrane fluidity can affect PMNL chemotaxis by modulating the binding of a chemoattractant, FMLP, at the specific receptors (Yuli, Tomo-
membrane fluidity
polymorphonuclear
naga & Snyderman, 1982; Valentino et al., 1986; Yasui et al., 1988a, 1988b). Furthermore, several investigators have provided evidence for changes in fluidity of PMNL membrane following stimulation, e.g. phagocytosis (Ingraham et al., 1981), chemoattractants (Bultmann et al., 1984; Masuda et al., 1990). The aim of this study was to examine whether the impaired PMNL chemotaxis in younger children and its improvement with age are associated with a change in the PMNL membrane
fluidity. MATERIALS AND METHODS PMNL preparation Heparinized (10 U/ml) cord blood samples were obtained from placentas immediately after delivery. The neonates were between 37 and 42 weeks of gestation, of appropriate size for gestational age (± 1 5 s.d.), and free of stress and asphyxia. Venous blood samples were also taken from normal infants, children and adults. Informed consent was obtained from the children's parents and the adult subjects. PMNL were separated by a modification of the method of Bbyum (1968). The cells were washed twice with Dulbecco's phosphate-buffered saline (pH 7.4) and resuspended at appropriate concentrations in RPMI 1640 with 10% fetal calf serum for motility assays and in the buffer for assay for membrane fluidity. Assay of motility PMNL motility was assayed using the under agarose method (Yasui et al., 1988a). N-formylmethionyl-leucyl-phenylalanine (FMLP; Sigma Chemical Co., St Louis, MO; 10-8M) was used
Correspondence: Atsushi Komiyama, Department of Paediatrics, Shinshu University School of Medicine, Asahi 3-1-1 Matsumoto 390, Japan.
156
Membrane fluidity of neutrophil
determination of the formation of excimers of fluorescent pyrene molecules. The rate of the complex (excimer) formation between a ground-state pyrene molecule and an excited molecule (monomer) is dependent on the translational diffusion rate of pyrene molecule which is incorporated into the cell membrane. Therefore the ratio of excimer: monomer formation yields an information about membrane fluidity (Soutar et al., 1974; Bultmann et al., 1984). Measurement of PMNL membrane fluidity was carried out according to the method described previously (Masuda et al., 1987, 1990). Briefly, PMNL were fluorescently labelled by incubation with 15 yIM pyrenedecanoic acid (Molecular Probes, Junction, OR) for 15 min at 250C in a water bath, and were applied to flow cytometry. The viability ofthese cells was > 95% as judged by trypan blue exclusion. Fluorescence intensities of monomer and eximer pyrenedecanoic acid were determined by flow cytometer (FACS III; Becton Dickinson, Sunnyvale, CA) equipped with 5-W argon ion laser (360 nm excitation, 20 mW output) and band-pass filters of 400 and 450 nm (70 nm bandwidth; Corion, Holliston, MA) Membrane fluidity was expressed as a fluorescence intensity ratio of excimer and monomer pyrenedecanoic acid ('E/
as a chemoattractant agent, the concentration being effective for maximal directed migration. After the incubation at 370C for 3 h, migration was evaluated by measurement of the linear distance in the direction of the chemoattractant (chemotaxis) compared with those of control medium (random migration). The values were represented as migration distance ( x 0 3 mm). Each experiment was done in duplicate. Assay of membrane fluidity Membrane fluidity can be determined by the excimer-forming lipid technique (Galla & Hartmann, 1980), which is based on the
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Statistical analysis Best-fit correlations were made using a LAND LSPM98 computer program. Differences were considered significant at P < 0-05.
Io0F
0 Neonates
Children
Infants (1-12 mo)
Adults
Children (6-12y)
(1-6y)
RESULTS
Fig. 1. Random migration of polymorphonuclear leucocytes (PMNL) as evaluated by the under agarose method. Migration values represent the migration distance under agarose ( x 0 3 mm). Mean (thick bar) and s.d. (thin bars). There was no significant difference between any pair of
PMNL random migration under agarose was normal in neonates, infants, and children aged 1-12 years, compared with adults (Fig. 1). Mean neonatal PMNL chemotaxis (unidirect
groups.
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An increase in polymorphonuclear leucocyte chemotaxis accompanied by a change in the membrane fluidity with age during childhood K. YASUI, M. MASUDA*, T. TSUNO, T. MATSUOKA, A. KOMIYAMA, T. AKABANE & K. MURATA* Department of Paediatrics, Shinshu University School of Medicine, Matsumoto, and * Department of Clinicolaboratory Medicine, Kansai Medical University, Osaka, Japan (Acceptedfor publication I February 1990)
SUMMARY Leucocyte membrane fluidity affects cell deformability as well as the accessibility of receptors and the degree of their exposure on the membrane. These effects modulate the subsequent cellular responsiveness. We have assayed membrane fluidity of polymorphonuclear leucocytes of children, using an excimer-forming lipid technique in flow cytometry, and evaluated its relation to their chemotaxis to a formyl peptide. We report that membrane fluidity and chemotaxis of polymorphonuclear leucocytes are increased with age. These findings may have important implications for the physiological processes in polymorphonuclear leucocyte motility during childhood. Keywords N-formylmethionyl-leucyl-phenylalanine leucocytes
The impaired chemotaxis of polymorphonuclear leucocytes (PMNL) in human newborns and infants is an important cause of increased morbidity and mortality due to bacterial infections (Klein et al., 1977; Laurenti et al., 1980). The mechanisms have been studied, but little is understood of their operation so far (Wilson, 1986; Hill, 1987). Miller (1978) has indicated that neonatal PMNL fail to deform normally following chemotactic factor stimulation. Erythrocytes from human neonates also differ from those of adults in having decreased filterability (Linderkamp, Hammer & Miller, 1986). Crespo, Bifano & Freedman (1988) found that erythrocytes from human neonates have decreased membrane lipid fluidity, which is related to increased cell rigidity and impaired deformability. Therefore, decreased filterability of neonatal PMNL may be explained at least in part by immaturity of the PMNL membrane. Repo et al. (1980) indicated that the impaired chemotaxis of neonatal PMNL was due not only to defective deformability but also to a defect in cellular responsiveness. The initiation of some PMNL responses, for example chemotaxis, is conducted via specific receptors on the membrane (Williams et al., 1977; Yasui et al., 1987). The membrane functional proteins, which include receptors, are also regulated by membrane fluidity (Galla & Hartmann, 1980). Manipulation of PMNL membrane fluidity can affect PMNL chemotaxis by modulating the binding of a chemoattractant, FMLP, at the specific receptors (Yuli, Tomo-
membrane fluidity
polymorphonuclear
naga & Snyderman, 1982; Valentino et al., 1986; Yasui et al., 1988a, 1988b). Furthermore, several investigators have provided evidence for changes in fluidity of PMNL membrane following stimulation, e.g. phagocytosis (Ingraham et al., 1981), chemoattractants (Bultmann et al., 1984; Masuda et al., 1990). The aim of this study was to examine whether the impaired PMNL chemotaxis in younger children and its improvement with age are associated with a change in the PMNL membrane
fluidity. MATERIALS AND METHODS PMNL preparation Heparinized (10 U/ml) cord blood samples were obtained from placentas immediately after delivery. The neonates were between 37 and 42 weeks of gestation, of appropriate size for gestational age (± 1 5 s.d.), and free of stress and asphyxia. Venous blood samples were also taken from normal infants, children and adults. Informed consent was obtained from the children's parents and the adult subjects. PMNL were separated by a modification of the method of Bbyum (1968). The cells were washed twice with Dulbecco's phosphate-buffered saline (pH 7.4) and resuspended at appropriate concentrations in RPMI 1640 with 10% fetal calf serum for motility assays and in the buffer for assay for membrane fluidity. Assay of motility PMNL motility was assayed using the under agarose method (Yasui et al., 1988a). N-formylmethionyl-leucyl-phenylalanine (FMLP; Sigma Chemical Co., St Louis, MO; 10-8M) was used
Correspondence: Atsushi Komiyama, Department of Paediatrics, Shinshu University School of Medicine, Asahi 3-1-1 Matsumoto 390, Japan.
156
Membrane fluidity of neutrophil
determination of the formation of excimers of fluorescent pyrene molecules. The rate of the complex (excimer) formation between a ground-state pyrene molecule and an excited molecule (monomer) is dependent on the translational diffusion rate of pyrene molecule which is incorporated into the cell membrane. Therefore the ratio of excimer: monomer formation yields an information about membrane fluidity (Soutar et al., 1974; Bultmann et al., 1984). Measurement of PMNL membrane fluidity was carried out according to the method described previously (Masuda et al., 1987, 1990). Briefly, PMNL were fluorescently labelled by incubation with 15 yIM pyrenedecanoic acid (Molecular Probes, Junction, OR) for 15 min at 250C in a water bath, and were applied to flow cytometry. The viability ofthese cells was > 95% as judged by trypan blue exclusion. Fluorescence intensities of monomer and eximer pyrenedecanoic acid were determined by flow cytometer (FACS III; Becton Dickinson, Sunnyvale, CA) equipped with 5-W argon ion laser (360 nm excitation, 20 mW output) and band-pass filters of 400 and 450 nm (70 nm bandwidth; Corion, Holliston, MA) Membrane fluidity was expressed as a fluorescence intensity ratio of excimer and monomer pyrenedecanoic acid ('E/
as a chemoattractant agent, the concentration being effective for maximal directed migration. After the incubation at 370C for 3 h, migration was evaluated by measurement of the linear distance in the direction of the chemoattractant (chemotaxis) compared with those of control medium (random migration). The values were represented as migration distance ( x 0 3 mm). Each experiment was done in duplicate. Assay of membrane fluidity Membrane fluidity can be determined by the excimer-forming lipid technique (Galla & Hartmann, 1980), which is based on the
5 0h
000
0*0 0
:0
0"
*to
@000
:0 .
00
00
.00
4-01
00*
.0
00
0
ax c
0
to
157
30H
IM) ratio), which was calculated by the following formula: channel number (450 nm) x PMNL count on each channel channel number (400 nm) x PMNL count on each channel The high IE/IM ratio indicates a high membrane fluidity.
0'
20 F-
Statistical analysis Best-fit correlations were made using a LAND LSPM98 computer program. Differences were considered significant at P < 0-05.
Io0F
0 Neonates
Children
Infants (1-12 mo)
Adults
Children (6-12y)
(1-6y)
RESULTS
Fig. 1. Random migration of polymorphonuclear leucocytes (PMNL) as evaluated by the under agarose method. Migration values represent the migration distance under agarose ( x 0 3 mm). Mean (thick bar) and s.d. (thin bars). There was no significant difference between any pair of
PMNL random migration under agarose was normal in neonates, infants, and children aged 1-12 years, compared with adults (Fig. 1). Mean neonatal PMNL chemotaxis (unidirect
groups.
0
00
14
0 o
13
0
so
0
0
000
000
121a}
00
c
II
0
co0
0
00
00 0
9
-
0 0
0
000
80o
0
y= 1-32. log x + 10-6 r-0-63 (P
