ARTICLE
Auteur(s) : Verena
Wiegering1, Matthias Eyrich1, Christian
Wunder2, Helga Günther2, Paul G
Schlegel1, Beate Winkler1
1Department of Pediatrics, Pediatric Hematology,
Oncology and Stem Cell Transplantation program, University
Children’s Hospital Wuerzburg, Germany
2Department of Anaesthesia, University
of Wuerzburg, Germany
accepté le 13 Mai 2009
T cells are important regulatory cells of the immune system.
Many of their functions are mediated by the expression and
secretion of cytokines [1]. An imbalance in cytokine production
profiles has been demonstrated in several clinical conditions and
in pathophysiological mechanism in certain immunological diseases,
for example acute GVHD, HIV and several autoimmune diseases [2-4].
Furthermore, there are several clinical studies which have
demonstrated that cord blood stem cell transplantation is
associated with a lower incidence or decreased severity of GVHD [1,
4, 5]. It has been hypothesized that one central reason for this
observation may be decreased cytokine expression in cord blood T
cells [6, 7].
However, data involving normal cytokine production profiles for
the neonatal period onwards in healthy infants, and for children
and adolescents are lacking. This information however, is important
not only for the further elucidation of normal, immunological
variations among the different age groups, but also for studies on
cytokine networks in pathological conditions in children with a
variety of autoimmune, infectious or transplant-associated
diseases.
Intracellular staining of cytokines using flow cytometry is a
method that allows the simultaneous staining of cytokines and
surface markers. It thus permits identification of cytokine
production in subpopulations without prior cell sorting and without
the use of clones at a single cell level [8-10]. Lymphocytes can be
subdivided into naïve and memory cells, the latter having been
primed by an antigenic stimulus [11]. These subpopulations can be
identified by their expression of variant CD45 isoforms. Naïve
cells carry the CD45RA isoform while memory cells express the
CD45RO epitope [12]. Memory cells are expected to mount a secondary
immune response to a previously encountered antigen faster and more
effectively than non-primed lymphocytes.
Our systematic analysis, from birth until adolescence, is the
first to include more than 100 healthy children, and provides an
insight into the development of cytokine expression in lymphocyte
subpopulations during childhood.
Donors and methods
Materials
Antibodies to the surface epitopes CD3 (clone UCHT1), CD4 (RPA-T4),
CD8 (RPA-T8), CD16 (3G8)/56 (B159), CD19 (HIB19), CD45RA (HI100),
CD45RO (UCHL1), Ki67 (B56), TGFβ (TB21), TNFα (Mab11), IFNγ (B27),
IL2 (MQ1-17H12) and IL4 (MP4-25D2) were all purchased from BD
(Heidelberg, Germany). Paraformaldehyde, PBS, saponin, PMA,
ionomycin and brefeldin, as well as HEPES buffer, were obtained
from Sigma (Taufkirchen, Germany). RPMI medium was a product of
Seromed biochrome (Berlin, Germany).
Methods
Heparinized blood was obtained from healthy children; 1 x
106 cells/µL were suspended in RPMI 1640 (with 2.0 g/L
NaHCO3) containing 10% fetal calf serum, 1% glutamine
and 1% penicillin/streptomycin. Cells either were left unstimulated
or were stimulated with PMA (10 ng/mL) and ionomycin (1 µM) for 24
h at 37°C and 5% CO2. In order to promote the
accumulation of de novo synthesised cytokines in the Golgi
apparatus of the synthesising cells, brefeldin was added to the
cells at a concentration of 2.5 µg/mL. After stimulation, cells
were processed as described by Jung et al. [8]. Briefly, cells were
harvested, washed once in HBSS, stained with surface markers for 10
min in a dark room at room temperature and then fixed for 10 min
with 4% paraformaldehyde. Cells were washed twice, resuspended in
saponin-buffer (HBSS containing 0.1% saponin and 0.001 M HEPES
buffer). Cells were stained with directly conjugated, anti-cytokine
antibodies. Cells were incubated for 20 min at 4°C in the
dark, washed once in saponin buffer and finally resuspended in
HBSS. Cytokine production was determined in CD3+,
CD8+, CD4+, CD45RA+ and
CD45RO+ cells using a BD four colour Calibur flow
cytometer. Several controls were performed to prove the specificity
of intracellular cytokine staining. Thresholds for cytokine
positivity were set using unstained cells as a control for
non-specific bonds. Results for cytokine positive cells (mean ± SD)
are expressed as a percentage of the respective subpopulation.
A minimum of 10 000 lymphocyte-gated events was acquired in
list mode and analysed with cell quest software (Becton Dickinson).
Events were gated on lymphocytes via forward and side scatter and
for CD3+, CD3+CD4+ and
CD3+CD8+cells or
CD3+/CD45RA+/ CD45RO- and
CD3+/CD45RA-/CD45RO+.
Statistical analysis
Students’ paired t test for mean differences was used to analyze
data for levels of statistical significance among the three age
groups. Correlation between surface isotope expression and cytokine
production was assessed using the Pearson correlation coefficient.
In all statistical applications p < 0.05 was considered
significant.
Patients
Inclusion criteria for this study were healthy children of
Caucasian origin who had no history of chronic disease, no family
history of immune-mediated disease, no sign of acute or chronic
infection and who had not received any medication within the
preceding week that could have influenced immune function. The
children were immunized according to the recommended immunisation
schedule for children of the German Standing Committee on
Vaccination (STIKO).
In total, 117 samples were obtained. Age groups were constructed
to represent age-related changes in cytokine expression. The
distribution in the groups was as follow: group 1 (cord blood): 25
samples (14 boys, 11 girls); group 2 (< 2 y): 21 samples (13
boys, 8 girls, median age 212 days [0.6 years]); group 3 (2-6 y):25
samples (14 boys, 11 girls, median age: 1420 days [3.9 years]);
group 4 (6-12 y): 23 samples (15 boys, 8 girls, median age: 2 777
days [7.6 years]); group 5 (12-18 y): 23 samples (12 boys, 11
girls, median age: 5076 days [13.9 years]).
The study was approved by the Human Subjects Committee of our
institution (Study # 133/04, 9/28/07, 11/25705, 12/13/04). Patients
and guardians participating in this study gave informed consent
according to institutional guidelines following the Declaration of
Helsinki.
Results
Cytokine-producing cells in CD4+
and CD8+ T cell subsets
To define normal ranges for intracellular cytokine expression in
T-cells in childhood, a total of 117 subjects were studied. The
median results are presented in table 1
for each age group. In cord blood, a large number of
CD3+, CD4+ and CD8+ cells produce
IL-2, whereas IFN-γ production was low in all cord blood T cells.
Except for absolute IL-2-production, there was an obvious general
trend towards increasing cytokine expression with increasing age.
We found a significant positive correlation index between age and
cytokine production for IFN-γ, TNF-α and IL-4 in
CD3+cells (figure 1), for TNF-α and
IFN-γ in CD8+cells (figure 2) and for IFNγ in
CD4+ cells. It is also worth noting that we found an
increasing population producing both IL-2 and IFN-γ at the single
cell level. The expression of TH2-cytokines such as IL-4 was
comparatively low and did not reach significance. Double staining
cytokine expression of IL-4 and TH1 cytokines remained stable and
low in the different age groups.
In summary, we found decreasing IL-2 expression in T cells (CD4
and CD8 subset), increasing IFN-γ and TNF-α production (figure 1,2) and stable
IL-4, Ki67 and TGF-β levels [data not shown], with increasing
age.
Table 1 Normal range of intracellular cytokine
production by T lymphocytes in different age groups. Results are
shown as mean of percentage cytokine-positive cells of the
respective subpopulation ± standard deviation. Absolute count shown
in […]
|
Cord blood (group 1; n = 25)
|
0-2 years (group 2; n = 21)
|
2-6 years (group 3; n = 25)
|
6-12 years (group 4; n = 23)
|
12-18 years (group 5; n = 23)
|
|
CD3 (% of total lymphocyte count)
|
55 ± 9
|
75 ± 8
|
68 ± 8
|
71 ± 8
|
71 ± 6
|
|
Lymphocyte (% and [µL] of total leukocyte count)
|
41 ± 5 [5 400]
|
47 ± 8 [4 100]
|
46 ± 6 [3 600]
|
40 ± 4 [2 400]
|
32 ± 5 [2 100]
|
|
28 ± 5 [1 520]
|
27 ± 8 [1 107]
|
33 ± 7 [1 180]
|
40 ± 7,5 [1 056]
|
44 ± 7.5 [924]
|
|
4 ± 1 [124]
|
15 ± 9.5 [615]
|
15 ± 5,5 [540]
|
22 ± 7.5 [528]
|
24 ± 6 [504]
|
|
7 ± 3.5 [217]
|
15 ± 6 [615]
|
21 ± 4.5 [756]
|
22 ± 8.5 [528]
|
32 ± 6.5 [672]
|
|
CD3 IL-4 [µL]
|
0,2 ± 0.5 [6]
|
0.7 ± 1 [28]
|
1 ± 1 [30]
|
1 ± 0.5 [24]
|
1 ± 1 [21]
|
|
CD4 IL-2
|
71 ± 12
|
14 ± 8.5
|
29 ± 9
|
26 ± 7
|
37 ± 10
|
|
CD4 IFN-γ
|
4 ± 3.5
|
8 ± 3
|
11 ± 6.5
|
13 ± 6
|
17 ± 7.5
|
|
CD4 TNF-α
|
4 ± 2
|
17 ± 3
|
22 ± 9
|
24 ± 6
|
25 ± 5
|
|
CD4 IL-4
|
0.6 ± 0.5
|
1 ± 1
|
1 ± 1
|
1 ± 2
|
1 ± 1
|
|
CD8 IL-2
|
50 ± 9
|
18 ± 12
|
18 ± 9
|
25 ± 8
|
27 ± 7.5
|
|
CD8 IFN-γ
|
9 ± 3
|
21 ± 10.5
|
13 ± 9
|
15 ± 7
|
22 ± 6.5
|
|
CD8 TNF-α
|
7 ± 4
|
21 ± 3.5
|
20 ± 7
|
26 ± 7.5
|
23 ± 6.5
|
|
CD8 IL-4
|
1 ± 1
|
1 ± 2.5
|
2 ± 1
|
1 ± 0.5
|
1 ± 1
|
Cytokine production profiles in CD45RO and CD45RA T
cells
Aware of the fact that CD45RA and CD45RO cells might produce
different cytokines, we directly analyzed the expression of CD45
isotypes. In all age groups, IL-2 was mainly produced by CD4 cells,
the relative contribution of naïve and memory cell population being
approximately equal. IFN-γ was mainly expressed by CD8CD45RO cells,
whereas TNF-α was mainly expressed in CD4CD45RO cells, and IL4
expression was similar in memory CD4 and CD8 cells. At birth, the
memory T cell subset is very rare, but with increasing age the
expression by CD45RO progressively and significantly increases so
that in adults the majority of T cells display a memory phenotype.
A significant positive correlation between CD45RO cells and
IFN-γ production was found in both CD4 and CD8 positive cells (not
shown).
Different cytokine expression with respect
to gender
In addition to age-related changes in cytokine expression during
childhood, we compared cytokine production in male and female
children. Careful attention was paid to the balance of females and
males in each group. Interestingly, we found higher IL-2 expression
(p < 0.04) in males in age groups 1, 2 and 3 (age < 6 y).
With increasing age, we detected a reverse ratio in females, with
increased values of IL-2 in group 4 (p < 0.01), and of IFN-γ in
group 5 (p < 0.04), compared to males.
Discussion
Intracellular cytokine detection by FACS analyses is increasingly
used as a human immune status indicator.
There are different studies that have looked at cytokines in
healthy, elderly people [13], in neonates [6, 14] as well as in
patients with underlying diseases, for example GVHD, autoimmune or
atopic conditions [1, 4, 5]. However, there are only a few studies,
with relatively small cohorts (n < 50), that took a closer look
at changes in cytokine expression during childhood [7, 15]. These
investigators found a trend to lower cytokine production in very
young children [6, 7, 15].
The method for induction of intracellular cytokine expression is
well known [8, 9]. Mascher [16] performed kinetic and distribution
studies in adults, which confirmed that stimulation between 20 and
24 h seemed to be a good time frame for obtaining a stable level of
Th1 and Th2 cytokines; furthermore they showed that cytokines are
mainly expressed by memory T cells.
As expected, we were able to demonstrate that neonates show,
almost exclusively, T cells with a naïve phenotype (data not shown
[17]), the expression of the memory phenotype (expression of
CD45R0) enhancing with increasing age. As most cytokines are
predominantly expressed by memory cells, we found higher cytokine
levels in older children [6, 18]. In neonates, the naïve T cell
predominantly produces IL-2, which is in accordance with the
existence of the naïve, Th0-Type T cell. This cell produces IL-2,
and, via an auto-stimulatory route, participates in the maturation,
differentiation and production of T cells.
With the exposure to antigens in the environment in early
infancy and childhood, as demonstrated by the typical viral
infections during this phase, or the production of antibodies
against antigens encountered through vaccination, the immune system
starts to build memory T and B cells. In infancy, while B cells
start producing antigen-specific antibodies and immunoglobulin
titers rise, the development of memory T cells is demonstrated by
an increasing expression of effector-T cell cytokines such as IFN-γ
and TNF-α. Also, during the production of T cells, the cells
develop either a Th1 phenotype (production of IFN-γ) or a Th2
phenotype (IL-4). As production of Th2-Type cytokines such as IL-4,
IL-5 or IL-10 is generally very low, we did not see any trend with
advancing age.
We cannot exclude an influence of breast feeding versus formula
diet in respect to cytokine expression, especially in the very
young children. However, we did not look at this and there are only
very few data regarding this topic [19, 20]. However, this might be
interesting for future investigations.
When analyzing intracellular cytokine staining after stimulation
with PMA, ionomycin and brefeldin A in pediatric patients, we
found a positive, significant correlation with age and increasing
IFN-γ values in both helper and cytotoxic T cells. This corresponds
with findings of increased Th1 levels in older children described
by Chipeta, and Hoffman [7, 15]. Even though Berdat [21] could not
confirm a significant increase in TNF-α serum levels, we were able
to demonstrate a significantly higher expression in CD3+
and CD8+ cells in older children by intracellular
cytokine staining. However, we could not find the correlation in
CD4+ cells described by Hoffmann [15].
The significance of the changes in the Th1-Th2 balance with age
remains unclear. A shift from a predominant Th1 level in young
individuals towards a Th2 cytokines in the elderly has been
recently described [18]. We found higher, absolute cytokine
expression in Th1 and Th2 cells generally in older children, with a
Th1 predominance.
Interestingly, girls older than 12 years showed a significantly
higher IFN-γ production than boys of the same age. In the same age
group, the higher prevalence of autoimmune disease in females is
well recognised. Since a predominance of Th1-cytokines has been
demonstrated for many autoimmune diseases [2, 3], it may be
hypothesized that the higher production of IFN-γ in females might
be a contributing, genetic factor.
In summary, the aim of this study was to establish valid
cytokine values for intracellular cytokine expression in healthy
children using a common, easily reproducible method. We were able
to demonstrate an association between age and cytokine expression,
and have established age-specific tables of normal cytokine
expression values.
Acknowledgments
We are grateful to all patients, and their parents or guardians,
who consented to participate in this study. We thank the technical
staff of the stem cell processing unit (B.Vahsel, H. Tscherner) at
the University Children’s Hospital, Wuerzburg, for excellent
technical assistance. V. Wiegering is the recipient of a junior
investigator prize awarded by the South German Society of
Pediatrics (2007). This work was supported in part by a program
project grant (Z-2/7-28.06.04) IZKF Würzburg, and in part by a
training grant from the Tour of Hope Foundation (2006/2007).
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