Download Cord Blood Leucocyte Expression of Functionally Significant

Scand. J. Immunol. 53, 72±78, 2001
Cord Blood Leucocyte Expression of Functionally Significant
Molecules Involved in the Regulation of Cellular Immunity
S. HODGE,*² G. HODGE* & R. FLOWER² & P. HAN*
*Haematology Department, Women's and Children's Hospital, and ²University of South Australia, 72 King William Street, Adelaide, South Australia
(Received 12 July 2000; Accepted in revised form 20 October 2000)
Hodge S, Hodge G, Flower R, Han P. Cord Blood Leucocyte Expression of Functionally Significant
Molecules Involved in the Regulation of Cellular Immunity. Scand J Immunol 2001;53:72±78
The cellular immune system of the newborn infant is immature and hypo-responsive when compared with
adults. The extent to which immaturity of the leucocyte function underlies hyporesponsiveness in the
newborn is incompletely understood. In this study flow cytometric techniques were applied to investigate the
concurrent expression of a range of surface and intracellular leucocyte functional molecules and cytokines in
resting and stimulated cord and adult blood. Production of interleukin (IL)-2 and expression of the
components of its receptor, IL-2Ra/b/g, were investigated. No differences in the proportion of leucocytes
producing IL-2Ra and IL-2Rg were observed for newborns and adults. A lower proportion of T cells and
natural killer (NK) cells from newborns expressed IL-2Rb and upregulation of expression was slower. We
hypothesize that reduced IL-2Rb may curtail early autocrine IL-2 activation of immune responses in the
newborn. This hypothesis was supported by the observation that an increased proportion of stimulated T cells
from newborns produced IL-2 at 4 h poststimulation, but at 24 h the proportion was lower than for adult T
cells. The very low levels of interferon (IFN)-g produced by neonatal T cells and NK cells may also be partly
explained by a curtailment of early autocrine activation of T cells. Expression and kinetics of upregulation
for other functional molecules were studied. CD71, HLA-DR, tissue factor and CD152 levels were not
significantly different for adults and newborns, suggesting that cord blood leucocytes, in some respects, may
demonstrate functional maturity. IL-6 secretion by stimulated monocytes was also comparable in cord and
adult blood. However, IL-1a and IL-1b were produced by a lower proportion of monocytes from newborns
than adults. Similarly, tumour necrosis factor (TNF)-a production for monocytes and T cells was lower in
cord blood. The mean fluorescence intensity for IL-1a, IL-1b and TNF-a was also lower for leucocytes from
cord blood. These findings are significant in relation to the inability of newborn infants to mount a febrile
response to infection. The findings of lower expression of IL-2Rb and lower production of inflammatory
cytokines IL-1a, IL-1b and TNF-a is a basis for improved understanding of the immunological immaturity
of leucocytes in the newborn.
Dr S. Hodge, Haematology Department, Women's and Children's Hospital, 72 King William Street, North
Adelaide, South Australia 5006. E-mail: [email protected]
INTRODUCTION
The cellular immune system of the newborn infant is immature
and hyporesponsive in comparison to adults. Neonates are more
highly susceptible to infection than adults, and exhibit more
severe or prolonged symptoms when infected [1].
Lower leucocyte function in newborns when compared with
adults, especially impaired production of some cytokines, has
been widely reported [1±4]. IFN-g has been shown to be
q 2001 Blackwell Science Ltd
important for the regulation of immune responses. IFN-g
inhibits virus replication and growth of intracellular parasites
and enhances the phagocytic activity of macrophages [5, 6].
Immaturity of a newborn's ability to produce IFN-g would
therefore contribute to a functional deficiency of the immune
system.
The inability of the neonate to mount a febrile response to
infection is also thought to be a factor in the immunological
immaturity in the newborn. IL-1, IL-6 and TNF-a are involved
Markers of Cellular Immunity in Cord Blood 73
Table 1. MoAbs used for three colour analysis of surface and intracellular markers and intracellular cytokines
Panel of monoclonal antibodies
Cell type
T cell
Monocyte
NK cell
Control
FITC
PE
TNF-a
IFN-g
CD25
CD71
HLA-DR
CD122
i/c (BD)
i/c (BD)
s (BD)
s (D)
s (BD)
s (BD)
CD69
TNF-a
IL1-a
CD25
Tissue
factor
CD71
CD122
CD69
CD3
CD3
CD3
CD3
CD3
CD3
CD3
CD3
CD3
IgG1
PECy5
s (BD)
i/c (BD)
i/c (BD)
s (BD)
s (AD)
IL-2
CD69
CD25
CD132
CTLA-4
CD122
CD132
CD69
IL-6
IL1-b
CD25
CD132
i/c (BD)
s (BD)
i/c (BD)
s (P)
s (D)
i/c (P)
i/c (P)
i/c (BD)
i/c (BD)
i/c (BD)
i/c (BD)
s (P)
CD3 (IT)
CD3 (IT)
CD3 (IT)
CD3 (IT)
CD3 (IT)
CD3 (IT)
CD3 (IT)
CD3 (IT)
CD14 (IT)
CD14 (IT)
CD14 (IT)
CD14 (IT)
s
s
s
s
s
s
s
s
s
s
s
s
s
CD132
CD122
CD69
IFN-g
CD25
CD25
CD69
HLA-DR
CD122
CD122
CD132
CD132
IgG1
i/c (P)
i/c (P)
i/c (BD)
i/c (BD)
s (BD)
i/c (BD)
s (BD)
s (BD)
s (P)
i/c (P)
s (P)
i/c (P)
s (BD)
CD14 (IT)
CD14 (IT)
CD14 (IT)
CD56 (IT)
CD56 (IT)
CD56 (IT)
CD56 (IT)
CD56 (IT)
CD56 (IT)
CD56 (IT)
CD56 (IT)
CD56 (IT)
CD3, CD14 or CD45 (IT)
(D)
(BD)
(BD)
(BD)
(BD)
(BD)
(BD)
(BD)
(BD)
(BD)
(BD)
(BD)
(BD)
s, antibody used for surface staining; i/c, antibody used for intracellular staining; BD, Becton Dickenson; P,
Pharmingen; IT, Immunotech; D, Dako; AD, AmericanDignostica.
in the induction of fever and the acute phase response [7]. IL-6
is a cytokine produced primarily by monocytes and macrophages in response to stimulation by IL-1 [8]. Conflicting data
in relation to IL-6 expression by resting and stimulated cord and
adult leucocytes have been reported. Schibler et al. [9] reported
a lower IL-6 production by neonates when compared with adult
production. At birth, normal production of IL-6 in response to
stimuli has also been reported [2, 10].
The kinetics of the production of IL-1 by leucocytes from
adults and newborn babies has been reported to be similar [2, 7,
9±12], although the expression was significantly lower for
leucocytes from cord blood. Reports of levels of expression of
TNF-a by cord blood T cells have also contained conflicting
data [3, 7, 13]. An investigation of levels of production of
various cytokines by stimulated cord blood leucocytes may
clarify differences in leucocytes in the newborn that relate to
their inability to mount a febrile response to infection.
There have also been reports of significantly lower expression
of some cell-surface molecules important for the regulation of
leucocyte function, when compared with levels expressed in
adults [14]. Zola et al. [14] reported a significantly lower
expression of the b and g chains of the IL-2 receptor on the
surface of resting cord blood leucocytes than for adult
leucocytes, and reduced IL-2 dependent T-cell activation in
vitro. Zola et al. [14] suggested that, in the newborn, the IL-2
receptor may display a `functional immaturity', and therefore,
fail to mediate the transduction of the ligand-induced signal.
Other cytokine receptors also may not be fully functional in
newborns, resulting in a lack of mature function of some
immunological pathways.
The extent to which immaturity of leucocyte function
underlies immunological hyporesponsiveness in the newborn
is incompletely understood. We have applied flow cytometric
techniques to investigate concurrent expression of a range of
surface and intracellular leucocyte activation markers and
cytokines in resting and stimulated cord blood. Concurrent
surface and intracellular expression of functional molecules for
a variety of cell types has been investigated in the newborn. The
data presented further elucidates the basis of immunological
immaturity in the newborn infant.
q 2001 Blackwell Science Ltd, Scandinavian Journal of Immunology, 53, 72±78
74 S. Hodge et al.
Table 2. Cytokine production by stimulated cord and adult whole blood. The percentage of cells producing cytokine are shown, with the MFI at 24 h
shown in brackets
Adult
0h
4h
24 h
(5.8)
Cord
0h
4h
24 h
T-cell
IFN-g
IL-2
TNF-a
Monocyte
TNF-a
IL-1a
IL-1b
IL-6
NK cell
IFN-g
0.8
12.3
20.4 ^ 10.7
(7.6)
3.8
11
9.9 ^ 6.6
(7.7)
0.7
20.7
8.5 ^ 13.3
(10.4)
0.9
12.4
61.6 ^ 19.0
(15.4)
2.9
30.8
74.3 ^ 22.5
(22.3)
6
50
58.1 ^ 20.4
(6.0)
4.0
22.0
30.0 ^ 9.9
(2.7)
1.6
5.7
15 ^ 8.4
2.1
2.5*
2.0* ^ 2.0
(0.8)***
1.6
18**
3.5* ^ 6.5
(2.8)***
0.6
14*
4* ^ 6.7
(3.8)***
2.1
9.2
32.0* ^ 4.0
(3.1)***
2.8
35.9
52.0* ^ 13.0
(10.1)**
2.3
34.9
45* ^ 16.0
(12.6)***
4.0
14.0
25.0 ^ 10.2
(6.3)
0.9
0.5*
0.5** ^ 0.2
(0.1)***
Results expressed as mean of five experiments. Standard deviations did not change markedly with time, therefore SD shown for 24 h experiments
only.
* Significantly less proportion of cord cells expressing cytokine at specified time (P , 0.05).
** Significantly higher proportion of cord cells expressing cytokine at specified time (P , 0.05).
*** Significantly lower MFI for cord cells (i.e. amount of cytokine produced) at 24 h (P , 0.05).
MATERIALS AND METHODS
Collection and stimulation of whole blood
For investigation of the expression of functional molecules for
unstimulated leucocytes, blood from 25 healthy adults, or cord blood
from 25 normal deliveries, was collected into sodium heparin (20 U/ml,
Faulding, Adelaide, Australia) and processed within 3 h of collection.
For investigation of the kinetics of expression of surface and
intracellular functional molecules for stimulated leucocytes, blood from
a healthy adult, or cord blood from a normal delivery, was collected into
sodium heparin (20 U/ml, Faulding) and processed within 3 h of
collection. Five experiments were carried out on separate days using
cord and adult blood, stimulated and tested in parallel. Before
stimulation, whole blood was diluted 1 : 2 with RPMI. Incubation
was carried out at 37 8C in 5% CO2-in-air, and testing carried out at 0, 4,
and 24 h for T cells and 0, 4, 24 and 48 h for monocytes and NK cells,
as previously described [15]. Stimuli for investigation of expression of
functional molecules were: SEB (Sigma, St. Louis, MO, USA) (10 mg/ml),
E. coli lipopolysaccharide (LPS, Sigma) (100 ng/ml) as well as IL-12 (R &
D Systems Inc., Minneapolis, USA) (50 ng/ml) and PHA (Wellcome,
Beckenham, UK) (5 mg/ml), CD28 (Becton Dickinson, San Jose, CA,
USA) (10 mg/ml) was used as a comitogen with SEB in this study.
Stimuli for investigation of cytokine production by T cells were PMA
(Sigma) (25 ng/ml) plus Ionomycin (Sigma) (1 mg/ml). The stimuli for
investigation of cytokine production by monocytes and NK cells were
the same as those described above. The concentrations of stimuli were
previously found to be optimal for the various cell types (data not shown).
Staining of surface markers on leucocytes. Staining of surface
markers was carried out using the `whole blood lysis' method [16].
Table 1 presents the combinations of monoclonal antibodies (MoAbs)
used in this study, and the corresponding fluorescent dyes. A 5-ml
volume of the MoAb was added to 100 ml of whole blood mixture then
incubated in the dark at room temperature, for 15 min. In order to lyse
red blood cells, 2 ml FACSlyse (Becton-Dickinson) was added for
exactly 10 min at room temperature in the dark. The cells were then
centrifuged for 5 min at 500 g, the supernatant discarded, and the
cells washed once in 2 ml of 1% bovine serum albumin (BSA) (Sigma)
in Isoton 11 (Coulter Electronics, Hialeah, FL, USA). The cell pellet
was resuspended in 0.3 ml of 1% paraformaldehyde in Isoton 11. The
suspensions of fixed cells were kept at 4 8C in the dark and analyzed
within 24 h on a fluorescent activated cell scan (FACScan, Becton
Dickinson) using Lysis II software (Becton-Dickinson).
Staining of intracellular cytokines and intracellular functional
molecules. Staining of surface markers with fluorescent-conjugated
MoAbs was carried out immediately prior to staining of intracellular
cytokines, as described above, for identification of cell subsets. To
block Fc receptors and reduce nonspecific staining, 20 ml human Ig
(Intragam, CSL, Sydney, Australia) was added to each tube for 10 min
at room temperature. In addition, 50 ml of cold 20 mm EDTA (Sigma)
was added to each tube to prevent loss of monocytes by sticking and
clumping. This step was incorporated into the procedure before surface
staining and also after permeabilization of the leucocytes. For
intracellular staining, following the above processing, cells were
permeabilized with 500 ml FACSpermTM (Becton-Dickinson) for
10 min at room temperature, then washed with 0.5% BSA in Isoton
11, centrifuged at 500 g, and the supernatant discarded. The cells
were then incubated at room temperature for 30 min with an appropriate
fluorescent-conjugated antibody to the intracellular marker of interest.
After a further wash, the cells were resuspended in 1% paraformaldehyde in Isoton 11. For investigation of the cytokine production,
activation was carried out in the presence of monensin (Sigma) which
acted as a `golgi block', inhibiting intracellular transport, and retaining
the cytokines produced during activation inside the cell. For
simultaneous analysis of surface and intracellular functional molecules
(i.e. IL-2Ra/b/g), the `golgi block' was omitted and the percentage of
positive staining cells calculated as previously described [17]. The
MoAbs were obtained commercially, conjugated to fluorescein
q 2001 Blackwell Science Ltd, Scandinavian Journal of Immunology, 53, 72±78
Markers of Cellular Immunity in Cord Blood 75
Fig. 1. Representative experiment showing
kinetics of cytokine expression by: (A) T
cells from adult whole blood stimulated
with PMA 1 ionomycin for 24 h; (B) T
cells from cord blood; (C) monocytes from
adult blood stimulated with LPS for 48 h;
(D) monocytes from cord blood; and (E) T
cells and NK cells from adult (solid line)
and cord blood (broken line).
RESULTS
contrast to the changes observed for stimulated adult whole
blood at 4 h and 24 h (Fig. 1C).
The mean fluorescence intensity (MFI), a measure of the
amount of cytokine produced per cell, was investigated.
Significantly higher levels of production of IFN-g were found
for adult CD31 T cells and CD561 CD3±NK cells in
comparison to levels in cord cells. IL-1a, IL-1b and TNF-a
production by monocytes from stimulated adult blood was also
significantly higher than for cord blood cells (Table 2).
Inflammatory cytokine production
Expression of IL-2 receptor components
A summary of the production of various cytokines by adult and
cord blood leucocytes is presented in Table 2. A significantly
higher proportion of LPS-stimulated monocytes from adult
blood produced IL-1a, IL-1b and TNF-a when compared with
monocytes from cord blood (P , 0.05). The proportion of cord
blood and adult blood monocytes expressing IL-6 was not
significantly different at 4 h or 24 h (Fig. 1C, D).
The percentage of cord blood T cells in which production of
IL-2 was detected was significantly greater than adult T-cell
expression of this cytokine at 4 h (P , 0.05). At 24 h, however,
a significantly greater proportion of adult T cells than cord cells
expressed IL-2 (Fig. 1B).
The proportion of cord blood NK cells or T cells in which the
production of IFN-g was detected was not significantly
upregulated by stimulation with IL-12 and PHA. This was in
Levels of surface and intracellular expression for the three
known IL-2R components (IL-2Ra/b/g), for resting T cells,
monocytes and NK cells from adult and cord blood was
compared by testing 25 healthy adults and cord blood from 25
uninfected newborn babies. A summary of data for IL-2Ra/b/g
expression for resting adult and cord whole blood is presented in
Table 3. A higher proportion of T cells from adults expressed
IL-2Rb (CD122), both on the cell surface and intracellularly,
when compared to T cells from cord blood (surface expression
8.2% (adult) in contrast to 6.1% (cord): intracellular expression
12.5% (adult) in contrast to 6.1% (cord).
A significantly higher proportion of NK cells from adult
blood expressed IL-2Rb (CD122) on the cell surface when
compared to NK cells from cord blood (21.6% (adult) cf 15.4%
(cord): P ˆ 0.048).
isothiocyanate (FITC), phycoerythrin (PE), PerCP or Cy5. Different cell
types were identified using third colour labelled MoAbs directed against
CD3, CD4, CD56 or CD14. In Table 1 the combinations of MoAbs to
intracellular cytokines used in the study are presented.
Statistical analysis. Differences between the median values for paired
data were tested using non-parametric techniques (Wilcoxon's test for
paired data).
q 2001 Blackwell Science Ltd, Scandinavian Journal of Immunology, 53, 72±78
76 S. Hodge et al.
Table 3. Expression of leucocyte activation markers by resting adult
and cord whole blood leucocytes. Results expressed as mean ^ SD from
testing of 25 uninfected adults and 25 uninfected newborns
Functional
Molecule
T cell
CD25
CD25i/c
CD69
CD69i/c
CD122
CD122i/c
CD132
CD132i/c
HLA-DR
CD152
CD71
Monocyte CD25
CD25i/c
CD69
CD69i/c
CD122
CD122i/c
CD132
CD132i/c
CD71
Tissue factor
NK cells CD25
CD25i/c
CD69
CD122
CD122i/c
CD132
CD132i/c
HLA-DR
Adult
Cord
(mean ^ SD) (mean ^ SD)
8.6
7.1
5.8
4.9
8.2
12.5
13.0
85.3
6.6
2.9
6.4
7.3
9.0
8.1
14.4
10.9
21.9
25.4
69.5
7.3
11.5
4.8
8.3
5.6
21.6
16.2
22.5
70.3
5.2
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
5.3
2.8
3.8
3.2
4.7
8.7
9.7
4.6
3.7
3.0
3.3
5.1
5.6
6.4
9.9
6.6
10.8
18.2
6.4
4.9
0.7
5.1
4.6
5.3
13.3
8.0
13.6
13.9
3.3
9.8
7.0
6.7
6.0
6.1
8.3
13.8
81.8
5.5
3.9
8.1
6.1
5.9
7.3
9.3
9.6
18.0
22.1
73.5
8.4
8.3
5.1
8.0
15.7
15.4
13.9
22.4
73.5
12
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
^
5.2
3.2
6.1
3.5
3.7
4.7
8.1
10.6
4.9
1.7
5.3
4.4
3.4
5.4
2.5
5.6
4.8
10.4
9.1
4.1
4.1
2.7
4.4
10.9
10.8
4.8
15.0
13.4
12.3
*P ˆ 0.046
*P ˆ 0.048
CD71, HLA-DR and CTLA-4) was investigated and compared
for cord and adult blood. A significantly higher proportion of
NK cells from cord blood expressed CD69 (both surface and
intracellular) when compared to NK cells from adult blood
15.7% (cord) compared with 5.6% (adult): P , 0.05). For
stimulated cord and adult blood no significant differences in the
kinetics of expression for HLA-DR, CD71 or CTLA-4 were
detected. (Wilcoxon test for paired data, P . 0.094 for all
analyses. Data not shown).
Despite the significantly higher proportion of NK cells from
cord blood expressing CD69, a different kinetics of expression
of CD69 was observed. Up-regulation of CD69 expression by
cord blood NK cells was slower than for NK cells from adult
blood (24 h for cord NK cells compared with 4 h for NK cells
from adults).
DISCUSSION
²P ˆ 0.004
*P ˆ 0.048
* Marker expressed by significantly smaller proportion of resting
cord blood leucocytes (P , 0.05).
² Marker expressed by significantly greater proportion of resting
cord blood leucocytes (P , 0.05).
For IL-2Ra (CD25) and IL-2Rg (CD132), no significant
differences in levels of surface or intracellular expression were
observed between adult and cord blood for any of the cell types
tested.
The kinetics of expression of IL-2Ra (CD25), IL-2Rb
(CD122) and IL-2Rg (CD132) was investigated for cord and
adult blood, after stimulation for 48 h. No significant differences in the kinetics of expression for IL-2Ra/b/g were detected
for cord and adult blood. (Wilcoxon test for paired data,
P . 0.094 for all analyses. Data not shown).
Expression of other leucocyte functional molecules
Expression of additional functionally significant molecules
involved in cell activation and signal transduction (CD69,
Neonates are more highly susceptible to infection than adults.
Part of the reason for this susceptibility is believed to be the
immunological immaturity of leucocytes in the newborn.
In this study a whole blood, flow cytometric assay was
applied for investigation of the kinetics of surface and
intracellular expression of functional molecules for T-cells,
NK cells and monocytes from heterogenous cells populations in
adult and cord blood. Flow cytometry has limited sensitivity
when compared with some other techniques (1000±2000
receptor molecules detection limit). The technique, however,
is sensitive enough to allow for comparative study of expression
of functionally important molecules from resting and activated
leucocytes in heterogenous cell populations from whole blood (a
mode which reflects the complexity of the in vivo environment
more closely). Furthermore, the use of a whole blood assay
minimises handling and possible artefactual activation of cells
which may occur during cell separation procedures. Using these
techniques, we found some notable differences between cord
blood leucocytes and adult leucocytes, including differences in
the profile of cytokines produced and response to stimuli.
No significant differences in the proportions of adult or cord
leucocytes expressing IL-2Ra or IL-2Rg were detected.
Detectable levels of surface and intracellular IL-2Rb were
expressed by a significantly lower proportion of newborn T cells
than adult T cells. A lower proportion of newborn NK cells,
compared with adult cells expressed surface IL-2Rb. The
kinetics of upregulation of the expression of IL-2Rb over 48 h
was similar for cord and adult leucocytes. In various studies, the
signal transduction function has been shown to be associated
with the b chain of the IL-2R on T cells [18, 19]. We
hypothesize that the low proportion of cord T cells expressing
IL-2Rb may play a role in the relative inability of leucocytes
from the newborn to produce IFN-g, by slowing assembly of
the high affinity IL-2R. This would restrict early transduction of
IL-2R ligand-induced signalling.
Slow assembly of the high affinity IL-2R may curtail early
autocrine IL-2 activation of T cells in the newborn. This
q 2001 Blackwell Science Ltd, Scandinavian Journal of Immunology, 53, 72±78
Markers of Cellular Immunity in Cord Blood 77
hypothesis is supported by our observation that an increased
proportion of stimulated T cells from newborns, compared with
adult T cells, produced IL-2 at 4 h, however a reduced
proportion of these cells produced IL-2 at 24 h.
The data presented in this study supports our previous report
[20] that a negligible proportion of stimulated T cells and NK
cells from newborns produced IFN-g when compared with the
production in adults. The IFN-g production by NK cells is
essential in the prevention of infection by intracellular microbial
pathogens [21], possibly by the contribution of IFN-g to
development of a Th1 type response [6]. The lower levels of
IFN-g production by neonatal T cells and NK cells may
therefore partly explain the high susceptibility of newborns to
fungal, viral, protozoan and certain bacterial infections.
IL-2 is required to maintain a high level of IFN-g production
by in vivo primed CD81 cells in viral infections [22]. In this
study we found a lower production of IL-2 by neonatal CD41
cells when compared with adult cells. The low rate of
production may reduce continued cytokine release by antiviral
CD81 cells in the newborn.
The rates of expression of various activation markers by
leucocytes were compared for 48 h in adult and cord blood after
incubation with various stimulating agents. CD69 was expressed
on a higher proportion of resting cord blood NK cells than
peripheral blood NK cells from adults. Despite this finding,
delayed upregulation of the proportion of stimulated cord blood
NK cells expressing CD69 was observed when compared with
adult cells (24 h compared to 4 h for NK cells from adults). It
has been suggested that CD69 has a functional role in redirected
lysis, mediated by activated NK cells [23]. The delay in
upregulation of CD69 may therefore indicate that the development of cytolytic action of NK cells from newborns may be
slower than in adults following an infective stimulus.
Functional maturity of leucocytes from newborns was
investigated by the expression of markers which are involved
in antigen presentation (HLA-DR), regulation of cell proliferation (CD71), T-cell costimulation (CD152) or coagulation
activation (tissue factor). The expression of these markers or
their kinetics of the upregulation, was not significantly different
for adults and newborns. These data indicate that leucocytes
from newborns may not be functionally immature in all areas.
For both adult and cord blood monocytes, the expression of
CD71 was upregulated earlier on stimulated monocytes rather
than T cells. The interaction of IL-2 with its high affinity
receptor is an essential requirement for T cells to express
transferrin receptor and to start proliferating [24]. That the high
affinity IL-2R is potentially expressed at a later time point on
stimulated T cells rather than monocytes has been reported [17].
Delayed expression of the high affinity IL-2R on T cells may
explain the delayed upregulation of CD71 and may be a basis of
delay in the proliferation of T cells when compared with
monocytes in an inflammatory response.
IL-1, IL-6 and TNF-a are involved in the induction of fever
and the acute phase response [7]. In this study the IL-6 secretion,
by LPS-stimulated monocytes, was compared in adult and
neonatal blood suggesting that the production of this cytokine in
response to the stimuli was normal at birth. The data on the
expression of IL-6 is in agreement with other reports [2, 10] but
is in contrast to data in some other studies [9]. Schibler et al. [9]
reported that only half as much IL-6 was produced from
peripheral blood mononuclear cells (PBMC's) from five fullterm neonates, when compared to those from five adults.
Schibler et al. [9] further reported that the peak production was
even lower for monocytes from preterm infants.
IL-1a and IL-1b were expressed by a significantly lower
proportion of monocytes from cord blood at 4 h and 24 h
following stimulation, when compared with expression by adult
monocytes, although the kinetics of upregulation was similar to
that for adults. These findings are consistent with those
described elsewhere [2, 11, 12].
There have been conflicting reports in relation to the levels of
expression of TNF-a for cord blood T cells [3, 7, 13]. In this
study, a reduced proportion of stimulated monocytes and T cells
from cord blood produced TNF-a when compared to the
proportion for stimulated adult cells. The intensity of fluorescence,
reflecting the quantity of cytokine produced per cell, was also
significantly reduced. It is likely that the basis of the failure of
neonates to mount a febrile response to infection is multifactorial
with reduced IL1 and TNF-a production playing some part.
For cord leucocytes, activation was observed but with a lower
proportion of cells expressing the b chain of the IL-2R (CD122)
and inflammatory cytokines IL-1a, IL-1b and TNF-a. A
delayed expression of CD69 on NK cells was also observed.
The data presented in this report provide a basis for further
understanding of the immunological immaturity of leucocytes in
newborn infants.
R E F E RE N C E S
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