doi:10.1006/cyto.2000.0706, available online at http://www.idealibrary.com on
SHORT COMMUNICATION
WEANING INDUCES AN INCREASE IN THE
NUMBER OF SPECIFIC CYTOKINE-SECRETING
INTESTINAL LYMPHOCYTES IN MICE
E. Vázquez,1,2 A. Gil,2 E. Garcı́a-Olivares,2 R. Rueda1
Intestinal immunity differs from systemic immunity in several aspects and is frequently studied
separately. In this work we have analysed the frequency of mononuclear cells spontaneously
secreting the cytokines IL-2, IL-4, IL-5, IL-6, IL-10, interferon gamma (IFN-) and tumour
necrosis factor (TNF-), in Peyer’s patches and lamina propria of small intestine in mice by
enzyme linked immunosorbent spot (ELISPOT) during 1 month after weaning. We have found
a high percentage of spontaneous Th1 as well as Th2 cytokine-secreting lymphocytes in both
populations, Peyer’s patches and lamina propria. An increase in the number of the lymphocytes
secreting most of the studied cytokines, at 1 and 2 weeks after weaning, was also observed. These
results suggest that the increase in the number of cytokine secreting lymphocytes may be one of
the potential mechanisms involved in the development of the intestinal immune system at
weaning.
2000 Academic Press
The lymphocytes lining the gut are able to defend
the organism against bacterial infection and participate
in the development of oral tolerance to food antigen.1
The lamina propria lymphocytes (LPL) are phenotypically similar to peripheral blood lymphocytes
(PBL)2 and functionally the intestinal lamina propria is
a major mucosal effector site. The B-lineage cells
represent 20–40% of LPL but there are also a substantial number of T cells (nearly 50%).3 The Peyer’s
patches are germinative centres and their predominant
role is to co-operate in the differentiation of B cells
towards IgA-producing cells.4 In this process T cells,
predominately CD4 + , are involved.5
In the gut, there is a wide spectrum of processes
for which cytokines play a relevant role; among the
total pool of intestinal cytokines, IL-2, IL5, and IL-6
have been shown to promote the maturation of murine
From the 1R&D Department, Abbott Laboratories S.A., Granada,
Spain; 2Department of Biochemistry and Molecular Biology,
University of Granada, Granada, Spain
Correspondence to: Enrique Vázquez Hernández, Abbott
Laboratories R&D Department, Cno/ Purchil no. 68, C.P.:
18004, Granada, Spain
Received 18 October 1999; received in revised form 21 February
2000; accepted for publication 5 April 2000
2000 Academic Press
1043–4666/00/081267+04 $35.00/0
KEY WORDS: weaning/cytokine/ELISPOT/intestinal lymphocytes/
mice
CYTOKINE, Vol. 12, No. 8 (August), 2000: pp 1267–1270
IgA-expressing cells to IgA-secreting cells. Thus,
cytokines from Th1 and Th2 populations influence the
differentiation of murine IgA-expressing cells.6 Since
cytokines are involved in processes of cell maturation,
there should probably be a high production and
secretion of cytokines during the first stage of life in
maturing and differentiating organs such as the small
intestine.
The aim of this study has been to assess the
number of IL-2, IL-4 , IL-5, IL-6, IL-10, IFN- and
TNF- secreting cells in Peyer’s patch lymphocytes
(PPL) and LPL of mice for one month after weaning.
We have found high proportion of cytokine-secreting
cells in both LPL and PPL of weanling mice, suggesting that other cells, different than CD4+ cells, may be
involved in the process of cytokine secretion at the
intestine.
RESULTS
Table 1 shows the mean percentages for IL-2-,
IL-4-, IL-5-, IL-6-, IL-10-, IFN- and TNF--secreting
lymphocytes in LPL and PPL of mice on days 3, 7, 14
and 30 after weaning. Both populations, LPL and PPL,
showed a very similar behaviour regarding the secreted
cytokine profile. The number of cytokine-secreting
lymphocytes was relatively higher, except for IL-4 and
1267
1268 / Vázquez et al.
CYTOKINE, Vol. 12, No. 8 (August, 2000: 1267–1270)
TABLE 1. Mean percentages of cytokine-secreting cells in intestinal lymphocytes populations, Peyer’s patches and lamina propria,
at 3, 7, 14 and 30 days after weaning
Cytokines
IL-2
IL-4
IL-5
IL-6
IL-10
IFN-
TNF-
Peyer’s patches
3 days
7 days
14 days
30 days
5.71.53
17.11.56
12.92.46
13.57.97
a
c
b
b
2.30.98
1.20.66
3.50.71
1.60.60
ab
a
b
a
7.51.94
22.02.41
22.21.25
3.92.00
b
c
c
a
17.02.45
25.32.37
24.21.87
14.64.82
a
b
b
a
10.71.35
26.09.79
24.57.98
19.04.84
a
c
c
b
11.32.18
35.86.32
19.01.96
3.51.19
b
d
c
a
1.80.49
1.00.47
1.80.33
0.10.06
b
b
b
a
Lamina propria
3 days
7 days
14 days
30 days
17.32.86
18.01.94
22.91.63
18.02.48
a
a
b
a
0.30.07
1.80.52
2.30.69
1.60.54
a
b
b
b
17.11.89
25.21.76
24.22.64
10.72.02
b
c
c
a
14.62.54
25.62.19
26.62.65
14.53.54
a
b
b
a
29.43.23
30.82.07
36.71.90
21.71.79
b
b
c
a
8.53.07
33.04.35
19.02.10
3.61.72
a
b
c
d
0.30.17
0.30.14
2.20.93
0.90.12
a
a
b
b
a<b<c<d. Values in the same column with a different letter were significantly different (P<0.05).
40
*
% Th1 and Th2 cells
30
*
20
*
*
10
0
3
7
14
30
Time after weaning (days)
Figure 1.
Percentages of Th1 ( ) and Th2 ( ) cells in small intestine Peyer’s patches total lymphocytes in mice at 3, 7, 14 and 30 days after weaning.
Bars show the mean percentagesstandard deviation of the Th1 (secreting IL-2 or IFN-) and Th2 (secreting IL-5 or IL-6) cytokine-secreting
cells from Peyer’s patches lymphocytes. (*) significant differences with respect to day 3.
TNF-. For most of the cytokines the percentage
of secreting-cells was significantly higher between the
days 7 and 14 compared with day 3, IFN- being the
cytokine with the strongest increase in both populations (LPL and PPL). After this peak, for almost all
the cytokines the percentages of secreting lymphocytes
decreased, being either significantly lower or equal
than the percentages on day 3, except for IL-2 and
IL-10 in PPL, for which they were significantly higher
on day 30 respect to day 3.
Figures 1 and 2 show Th1 (secreting IL-2 or
IFN-) and Th2 (secreting IL-5 or IL-6) lymphocytes in
both populations, PPL and LPL, respectively. Th1 and
Th2 cells exhibited a highly similar pattern in Peyer’s
patches as well as in lamina propria. There was a
strong increase of the cytokine-producing cells at days
7 and 14 respect to day 3 in both profiles. However, on
the following days, the values were decreased reaching
percentages similar to day 3 or even significantly lower
(Th1 profile in LPL)
DISCUSSION
One of the most important results we have
found is the high number of spontaneous cytokine
producing intestinal lymphocytes in mice at early
weaning. In other lymphoid organs different from the
intestine, the percentage of lymphocytes producing
cytokines is usually very low.7 However, in LPL,
as well as PPL, we have found a high percentage
of resting cytokine-producing lymphocytes. These
results agree with those obtained by other authors
Intestinal cytokines at weaning / 1269
40
*
% Th1 and Th2 cells
30
*
*
*
20
*
10
0
3
7
14
30
Time after weaning (days)
Figure 2.
Percentages of Th1 ( ) and Th2 ( ) cells in small intestine lamina propria total lymphocytes in mice at 3, 7, 14 and 30 days after weaning.
Bars show the mean percentagesstandard deviation of the Th1 (secreting IL-2 or IFN-) and Th2 (secreting IL-5 or IL-6) cytokine-secreting
cells from Peyer’s patches lymphocytes. (*) significant differences with respect to day 3.
which showed a high percentage of intestinal cytokine
producing cells in normal mice8 and humans.8,9
Our results suggest that in addition to CD4 + T
lymphocytes, other intestinal lymphocytes are able to
produce cytokines, although their potential functional
implication are not well known. The production of
cytokines by human10 and murine11 blood CD8 + T
lymphocytes has been previously described, demonstrating that both MHC class I- and class II-restricted
T cells has the potential to regulate immune responses
by secreting the same lymphokines. Likewise, Taguchi
et al.,12 have demonstrated that, in terms of cytokine
production, CD8 + TCR-cells have subsets similar to
those described for CD4 + Th cells.
Our results in Peyer’s patches are very similar
to those observed in lamina propria lymphocytes.
Likewise, in Peyer’s patches as well as in lamina
propria we could observe a generalized increase of
cytokine producing lymphocytes (IL-2, IL-5, IL-6,
IL-10 and IFN-) around 7 to 14 days after weaning.
These results suggest that the stimulation by food
antigens may induce a high production of cytokines by
intestinal lymphocytes, which could be involved in the
process of maturation and differentiation of the
immune system at Peyer’s patches and lamina propria.
Another remarkable aspect of our results is that
the cytokine profile observed in lamina propria and
Peyer’s patches is not characteristic of Th1 or Th2 cells,
but of a mixture of them. Taking into account that the
main process in the maturation of the intestinal
immune system is the generation of IgA-producing B
cells, both kind of cytokines (secreted by Th1 and Th2
subsets) might be involved in the promotion of that
physiological process, according to our results and
those obtained by other authors.6,13
In conclusion, the high production of cytokines by
intestinal lymphocytes could be one of the mechanisms
involved in the maturation and differentiation of the
intestinal immune system at weaning.
MATERIAL AND METHODS
BALB/c male mice (four weeks old) were purchased from
Iffa Credo, (France). Once mice arrived they were weighed
and housed under normal conditions Mice were sacrificed at 3,
7, 14 and 30 days after the beginning of the experiment. The
isolation of intestinal lamina propria lymphocytes and Peyer’s
patches lymphocytes was made following the procedure of
Gautreaux et al.14 The ELISPOT assay was carried out basically as described by Fujihashi et al.15 This method is designed
to detect specific cytokine-secreting cells at a single level. In
this work we have tested the frequency of IL-2-, IL-4-, IL-5-,
IL-6-, IL-10-, IFN- and TNF--producing lymphocytes in
LPL and PPL. The results were statistically analysed as follows: results were expressed as mean percentagesstandard
deviation. The homogeneity of variances was tested by
Levene’s test and one-way Anova. The Bonferroni test was
used to evaluate significant differences (P<0.05) between 3, 7,
14 and 30 days. All test were performed using the PC90
version of 7D BMDP programme (BMDP PC 90 version,
Statistical Software, Inc, Los Angeles, CA, USA).
Acknowledgements
We are gratefully indebted to Mrs Maria Luisa
Jiménez López for help and technical assistance.
1270 / Vázquez et al.
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