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A study of CD45RO, CD45RA and CD29 antigen expression on human decidual T cells in an early stage of pregnancy
Immunology Letters, 40 (1994) 193-197 Elsevier Science B.V.
IMLET 02103
A study of CD45RO, CD45RA and CD29 antigen expression on human decidual T cells in an early stage of pregnancy •
a
*
S. Salto ' , K . N i s h i k a w a b, T. M o r i i b, N. N a r i t a b, M . E n o m o t o a, A. I t o a a n d M . Ichijo a aDepartment of Obstetrics and Gynecology, Nara Medical University, Nara 634, Japan; and bSeeond Department of Internal Medicine, Nara Medical University, Nara 634, Japan (Received 10 February 1992; accepted 10 November 1992)
1.
Summary
The decidua is the place where the fertilized egg is implanted and where the immunocompetent cells of the mother come into direct contact with genetically disparate cells of the conceptus. Although the T cells in the decidua are exposed to fetal antigens, the fetus is not rejected by maternal immunocompetent cells. In the present study, we examined surface markers to determine whether the T cells in the human decidua are naive T cells without or memory T cells with a history of antigen stimulation. Although few T cells were present in the decidua, as compared to the peripheral blood, CD45RO ÷, CD29 ÷ and C D 4 5 R A CD4 ÷ T cells as well as CD45RO ÷, CD29 ÷ and C D 4 5 R A - CD8 ÷ T cells, which are considered to be memory T cells, were in the majority, with only small numbers of C D 4 5 R O - , CD29and CD45RA ÷ CD4 ÷ and CD8 ÷ cells, which are naive T cells, present. Also, the decidual mononuclear cells secreted IL-2 and IL-4. Since IL-4 is secreted only by memory T cells, it is suggested that in the decidua memory T cells increase in number and secrete cytokines, thereby in some way influencing the phenomenon of fertility.
other using CD45RA, CD45RB and CD45RO antibodies [3-5]. T cells stained with CD45RO antibody are regarded as memory T cells (T cells which have a history of activation by antigens) [6-10]. The decidua is the place where the fertilized egg is implanted and the placenta grows. Recent studies have suggested that maternal T cells play an important role in maintaining pregnancy in mice [11-15]• In these reports, soluble T-lymphocyte factors (i.e., GM-CSF and IL3) enhance the proliferation of murine trophoblast cells in vitro. Wegrnan [12] proposed that immunostimulation, rather than suppression, is important in maintaining pregnancy. If this model is valid in humans, T cells in the decidua should possess the CD45RO and CD29 antigens because many cytokines are secreted from memory T cells. In this study, using flow cytometry, we examined whether the ratio of memory T cells is increased in the human decidua. Furthermore cytokine secretion (IL-2 and IL-4) from decidual mononuclear cells was examined. 3. Materials and Methods
3.1.
Isolation of mononuclear cells from decidual tissue
2. Introduction
CD45 gene expression is found in almost all cells of the hemopoietic system [1,2]. Because of differences in m R N A splicing, there are isoforms of the CD45 gene that can be distinguished from each Key words: CD45RO; Decidua; T cell; Cytokine; Human and memory T cell *Corresponding author: Shigeru Saito, Dept. of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Kashihara-shi, Nara 634, Japan. SSDI 0 1 6 5 - 2 4 7 8 ( 9 3 ) 0 0 0 1 9 - N
Human decidual tissue samples from the early stages of pregnancy (weeks 6--11 of pregnancy) were cut into pieces and filtered through a nylon mesh (30 #1 in diameter). Then, by Ficoll-Hypaque gravity centrifugation, mononuclear cells were separated out. Prior to the study, informed consent was obtained from all subjects. 3.2.
Isolation of mononuclear cells from peripheral blood
Heparinized peripheral blood was subjected to Fi-
coll-Hypaque gravity m o n o n u c l e a r cells.
centrifugation
to
separate
4. Results
4.1. 3.3.
Flow cytometry
Phycoerythrin (PE)-labeled Leu-2 (CD8), PE-labeled Leu-3 (CD4) and fluorescein isothiocyanate (FITC)-labeled Leu-18 ( C D 4 5 R A ) were purchased from Becton Dickinson (USA). FITC-labeled U C H L - 1 ( C D 4 5 R O ) m o n o c l o n a l a n t i b o d y (mAb) was purchased f r o m Nichirei (Japan) and F I T C - l a beled 4B4 (CD29) was purchased f r o m Coulter (USA). Using a c o m b i n a t i o n o f PE-labeled and FITC-labeled mAbs, we conducted 2-color flow cytometry with an F A C S c a n (Becton Dickinson, USA).
3.4.
Determination o f lL-2 and IL-4
Decidual m o n o n u c l e a r cells were suspended in R P M I - 1 6 4 0 m e d i u m containing 10% fetal calf seru m at a concentration o f 1 x 106/ml, and 200 pl o f the suspension was placed into each well o f a 96-well microtiter plate. The supernatant was harvested 24 h later and stored at - 8 0 ° C . IL-2 was bioassayed with C T L L - 2 cells according to a previously reported m e t h o d [16]. The lowest detectable limit o f IL-2 in the samples o f the present study was 0.8 pM. IL-4 was assayed using an enzyme i m m u n o s o r b e n t assay kit (Amersham, UK). The lowest detectable limit o f IL-4 with this kit was 10 pg/ml.
3.5.
Expression of CD45RO, CD45RA and CD29 on CD4 + T-cell subset
O f peripheral blood lymphocytes (PBL) f r o m nonpregnant women, 41.1 ___8.1% (n = 10) were C D 4 +. O f C D 4 + cells, 44.0___7.4% (n = 1 0 ) showed C D 4 5 R O expression. O f P B L from 10 w o m e n in the early stages o f pregnancy, 36.9__ 6.8% were C D 4 + (Table 1). The percentage o f C D 4 ÷ cells a m o n g P B L did not differ between n o n - p r e g n a n t and pregnant women. O n the other hand, the percentage o f C D 4 5 R O ÷ cells a m o n g peripheral blood C D 4 + cells f r o m pregnant w o m e n was 5 5 . 5 _ 12.2%, which was significantly higher than the percentage for non-pregnant w o m e n ( P < 0 . 0 5 ) . O f decidual lymphocytes, 5 . 5 + 1.9% (n = 10) were C D 4 ÷. This percentage was a b o u t 1/7 o f the percentage for P B L from pregnant and n o n - p r e g n a n t women. The percentage o f C D 4 5 R O + cells a m o n g C D 4 + T cells from the decidua was 84.7___ 10.7%, which was significantly higher than the percentage for peripheral blood from n o n - p r e g n a n t and pregnant w o m e n ( P < 0 . 0 0 1 ) . We further analyzed the expression o f C D 4 5 R A and C D 2 9 on C D 4 + T-cell subset. As shown in Fig. 1, decidual C D 4 + cells bore high a m o u n t s o f C D 4 5 R O and C D 2 9 antigen, whereas decidual C D 4 + cells had low a m o u n t s o f C D 4 5 R A antigen. Identical findings were observed in two other individuals (data not shown).
Data analysis 4.2.
Values were expressed in mean__+SD. Student's t test was employed to test statistical significance.
Expression of CD45RO antigen on CD8 + T-cell subset
O f P B L from n o n - p r e g n a n t women, 24.2-t- 11.0% TABLE 1 CD45RO EXPRESSION ON DECIDUAL AND PERIPHERAL CD4 ÷ AND CD8 ÷ T CELLS All data indicate mean_ SD. CD4 + ( % ) a
CD45RO + CD4 + (%)a
CD45RO + CD4+/ CD4 + (%)b
CD8 + (%)a
CD45RO + CD8 + (%)a
CD45RO + CD8+/ CD8 + (%)¢
Non-pregnant women (PBL) n = 10
41.1 --- 8.1
17.8 + 2.8
44.0 - 7.4
24.2 + 4.8
6.3 ___2.2
27.1 + 11.0
Pregnant women (PBL) n = 10
36.9 __+6.8
20.2 + 5.0
55.5 + 12.2
24.8 + 5.9
8.5 + 3.9
34.3 + 14.3
5.5 ___ 1.9
4.5 _ 1.3
84.7 + 10.7
7.9 _ 5.6
4.3 + 2.5
58.4 ___ 13.2
Decidual lymphocyte n = 10
aData show the percentage of lymphocytes. bData show the percentage of CD45RO + CD4+/CD4 +. eData show the percentage of CD45RO + CD8+/CD8 +. 194
A. P B L (nonpregnant woman)
C. Decidual lymphocytes
B. P B L
(pregnant woman)
CD4
10 o
10 i
tO 2
. . . . . . .10 a
10°
IO'
i~12
1o
ta
l0 =
I0"
~ tO*
"';~ ' '"7,
IB
, 101
102
,,,,4 ........ l0 = 10'
CD 45 RO
o
A
. . . . . . . . . . . . . . . . . . . . . . i ...... t=l t(] 2 la ~ t
CD29
CD29
(3D 29
CD 45 RA
I0=
CD 45 RO
CD 45 RO
la I
CD4
CD 4
.... ';-",' "'T=,' '"';';~ ' '"';: CD 45 RA
............. ta I
I .............. 1o 2 1~ a
1
CD 45 RA
Fig. 1. CD45RO, CD29 and CD45RA expression on peripheral blood and decidual CD4 + T cells. PBL and decidual lymphocytes were stained with FITC-conjugated anti-CD45RO, CD29 and CD45RA and PE-conjugated anti-CD4. Sampleswere analyzed on a FACScan and displayed as histograms. The horizontal and vertical axes represent log fluorescence intensity and relative cell number, respectively.(upper column) CD45RO expression; (middle column) CD29 expression; and (lower column) CD45RA expression on CD4 ÷ cells. were CD8 + T cells. CD45RO was positive in 27.1 -t- 11.0% of CD8 + T cells. O f PBL from pregnant women, 2 4 . 8 + 5 . 9 % were CD8 + T cells and C D 4 5 R O was positive in 34.3___14.3% of CD8 + T cells (Table 1). The percentage of CD8 + T cells and the percentage of C D 4 5 R O + CD8 + T cells did not differ significantly between pregnant and non-pregnant women. On the other hand, the percentage of CD8 + cells among decidual lymphocytes was 7 . 9 + 5 . 6 % , which was about 1/4 of the percentage for PBL. The percentage of CD45RO ÷ cells among CD8 ÷ T cells from the decidua was 58.4+13.2%, which was significantly higher than the percentage for PBL from pregnant and non-pregnant women (P<0.001). In the decidua, most CD8 + T cells expressed CD29 antigens, whereas a few CD8 ÷ T cells expressed C D 4 5 R A antigens in all 3 cases (Fig. 2).
4.3.
IL-2 and IL-4 secretion of decidual mononuclear cells
IL-2 and IL-4 were identified in the supernatant of decidual mononuclear cells in 4 cases (Table 2). However, the levels of both cytokines in the supernatant of peripheral blood cells were below the detectable limit.
5. Discussion About 70-80% of h u m a n decidual lymphocytes are accounted for by C D 3 - C D 1 6 - CD56 bright natural killer cells called 'endometrial granulocytes' [1721]. The percentage of T cells in h u m a n decidual lymphocytes is lower than that in peripheral blood [1921]. Until now, very few reports on decidual T cells have been published. Recent reports suggested that maternal T cells play an important role in maintaining pregnancy in mice [11-15]. Bearing this in mind, we examined in the present study which phenotypes 195
A. P B L ( n o n p r e g n a n t woman)
B. PBL (pregnant woman)
CD 8
I0 °
I0'
CD8
I0= I0= 10"
I0 °
I0'
CD 45 RO
i~l I
15 3
I0 ~
10"
I~
11
% ~ , ,,,;~,, ,,,,;,;.,, ,,,,;;~,, ,,,;,, 4 CD 45 RA
CD 45 RA
I01
10 2
I0 ~
I0"
CD 45 RO
" , ' . ' "";,b' "",";~' "",'2~ ' '"';: CD29
.............. 1~] 2 '
10 2
CD8
CD 45 RO
" " " i ; ' " '"i;; -~'' '",';;~ .... CD29
~ ,,LI
C. Decidual lymphocytes
CD29
fll (
CD45 RA
Fig. 2. CD45RO, CD29 and CD45RA expression on peripheral blood and decidual CD8 + T cells. The horizontal and vertical axes represent log fluorescenceintensity and relative cell number, respectively.(upper column) CD45RO expression; (middle column) CD29 expression;and (lower column) CD45RA expression on CD8 + cells.
TABLE 2 IL-2 AND IL-4 PRODUCTION OF DECIDUAL MONONUCLEAR CELLS AND PERIPHERAL BLOOD MONONUCLEAR CELLSa Weeks of gestation
7 7 8 10
IL-2 (PM)
IL-4 (pg/ml)
decidua PBMCb
d e c i d u a PBMC
1.3 2.1 1.7 1.5
20 23 20 19
< 0.8 < 0.8 <0.8 <0.8
< 10 < 10 < 10 <10
aDecidual mononuclear cells and peripheral blood mononuclear cells were suspended in RPMI-1640 medium containing 10% fetal calf serum to a concentration of 1 x 106/ml. The cells were distributed in 0.2 ml aliquots into 96 round-bottomed wells and were cultured for 24 h. IL-2 concentration was determined by bioassay using IL-2-dependent CTLL-2 cells and IL-4 concentration was determined by EIA. bperipheral blood mononuclear cells. 196
o f T cells are present in the decidua (a place for imp l a n t a t i o n o f fertilized egg) primarily by analyzing the expression of C D 4 5 R O , C D 4 5 R A a n d CD29 antigen. I n this study, only a small n u m b e r of T cells was f o u n d in the decidua. However, most of the C D 4 + T cells a n d CD8 + T cells were m e m o r y cells which carried C D 4 5 R O a n d CD29 antigen a n d did n o t carry C D 4 5 R A antigen. C D 4 + m e m o r y T cells are reported to be a b u n d a n t in sites frequently exposed to recoil a n t i g e n (e.g., the skin, tracheal m u c o sa a n d intestinal mucosa) a n d in tissues affected by t u b e r c u l o u s pleurisy or arthritis [22-29]. I n these tissues, i m m u n o c o m p e t e n t cells are in an activated state. O u r previous study revealed that decidual T cells also express activation antigens such as CD69 a n d H L A - D R antigens [30]. It seems likely that the expression of C D 4 5 R O a n d CD29 antigens o n decidual T cells is the result of recognition of some fetal antigens by m a t e r n a l T cells. I n mice, M - C S F , G M C S F a n d IL-3 are reported to serve as placenta-proliferating factors [16]. Also in h u m a n s , M - C S F a n d IL-6 are reported to p r o m o t e the secretion a n d re-
lease o f h u m a n c h o r i o n i c g o n a d o t r o p i n f r o m the placenta [31,32]. C D 4 ÷ m e m o r y T cells are k n o w n to p r o d u c e large a m o u n t s o f c y t o k i n e s such as IL-2, IL-3, IL-4, IL-5, I L - 6 a n d I F N - ~ [33]. I n this study, we f o u n d t h a t dec i d u a l m o n o n u c l e a r cells secrete I L - 2 a n d IL-4. It has been r e p o r t e d t h a t IL-2 was p r o d u c e d by naive T cells a n d m e m o r y T cells [33]. H o w e v e r , I L - 4 was p r o d u c e d o n l y b y m e m o r y T cells [33]. These d a t a p r o v i d e evidence t h a t d e c i d u a l m e m o r y C D 4 ÷ T cells are i m m u n o c o m p e t e n t cells. It is highly p r o b able t h a t o t h e r c y t o k i n e s are p r o d u c e d by d e c i d u a l T cells a n d affect t r o p h o b l a s t g r o w t h o r differentiation. It was recently revealed t h a t C D 8 ÷ T cells also include m e m o r y T cells [9,10]. T h a t is, C D 4 5 R O ÷, C D 2 9 ÷ a n d C D 4 5 R A - C D 8 ÷ cells were recently f o u n d to possess a c y t o t o x i c activity. A l t h o u g h the significance o f an increase o f C D 8 ÷ m e m o r y cells in the d e c i d u a l tissue is u n k n o w n , this increase m a y be the result o f the sensitization o f the m a t e r n a l C D 8 T cells to some fetal cells. It is an o p e n q u e s t i o n w h e t h e r o t h e r c y t o k i n e s are p r o d u c e d b y d e c i d u a l C D 4 ÷ T cells a n d w h e t h e r C D 8 ÷ m e m o r y T cells exert a killer activity in the decidua.
References [1] Thomas, M.L. and Lefrancois, L. (1988) Immunol. Today 9, 320. [2] Thomas, M.L. (1989) Annu. Rev. Immunol 7, 339. [3] Hall, L.R., Steuli, M., Schlossman, S.F. and Saito, H. (1988) J. Immunol. 141, 2781. [4] Steuli, M., Morimoto, C., Schrieber, M, Schlossman, S.F. and Saito, H. (1988) J. Immunol. 141, 3910. [5] Thomas, M.L. and Lefrancois, L. (1988) Immunol. Today 9, 320. [6] Sanders, M.E., Makgoba, M.W., Sharrow, S.O., Stephany, D., Springer, T.A., Young, H.A. and Shaw, S. (1988) J. Immunol. 40, 1401. [7] Pulido, R. and Sanchez Madrid, F. (1989) J. lmmunol. 143, 1930. [8] Tedder, T.F., Clement, L.T. and Cooper, M.D. (1985) J. Immunol. 134, 2983.
[9] De Paoli, P., Battistin, S. and Santini, G.F. (1988) Clin. Immunol. Immunopathol. 48, 290. [10] Merkenschlager, M. and Beverley, P.C.L. (1989) Int. Immunol. 1,450. [11] Athanassakis, I., Blenchley, R.C., Paetkan, V., Guilbert, L., Barr, P.J. and Wegmann, T.G. (1987) J. Immunol. 138, 37. [12] Wegmann, T.G. (1988) Immunol. Lett. 17, 297. [13] Armstrong, D. and Chaout, G. (1989) Biol. Reprod. 40, 466. [14] Athanassakis, I., Chaout, G. and Wegrnann, T.G. (1990) Cell Immunol. 129, 13. [15] Chaouat, G., Menu, E., Clark, D.A., Dy, M., Minkowsky, M. and Wegrnann, T.G. (1990) J. Reprod. Fertil. 89, 447. [16] Saito, S., Saito, M., Kato, Y., Moriyama, I. and Ichijo, M. (1988) J. Reprod. lmmunol. 14, 247. [17] Hamperl, H. and HeUweg, G. (1958) Obstet. Gynecol. 2, 379. [18] Bulmer, J.N. and Johnson, P.M. (1985) Immunology 55, 35. [19] Starkey, P.M., Sargent, I.L. and Redman, C.W.G. (1988) Immunology 65, 129. [20] Nishikawa, K., Saito, S., Morii, T., Hamada, K., Ako, H., Narita, N., Ichijo, M., Kurahayashi, M. and Sugamura, K. (1991) Int. Immunol. 3, 743. [21] King, A. and Loke, Y.W. (1990) Am. J. Obstet. Gynecol. 162, 308. [22] James, S.P., Fiocchi, C., Graeff, A.S. and Strober (1986) Gastroenterology 91, 1483. [23] Bos, J.D., Hagenaars, C., Das, P.K., Krieg, S.R. and Voorn, W.J. (1989) Arch. Dermatol. Res. 281, 24. [24] Saltini, C., Kirby, M., Trapnell, B.C., Tamura, N. and Crystal, R.G. (1990) J. Exp. Med. 171, 1123. [25] Schieferdecker, H.L., Ullrich, R., Weiss-Breckwoldt, A.N., Schwarting, R., Stein, H., Riecken, E.O. and Zeitz, M. (1990) J. Immunol. 144, 2541. [26] Kingsley, C., Pitzalis, C., Kyriazis, N. and Panayi, G.S. (1988) Scand. J. Immunol. 28, 225. [27] Pitzalis, C., Kingsley, G., Haskard, D. and Panayi, G. (1988) Eur. J. Immunol. 18, 1397. [28] Lasky, H.P., Bauer, K. and Pope, R.M. (1988) Arthr. Rheum. 31, 52. [29] Choffion, M., Gonzalez, V., Weiner, H.L. and Hailer, D.A. (1989) Eur. J. Immunol. 19, 1791. [30] Saito, S., Nishikawa, K., Morii, T., Narita, N., Enomoto, M. and Ichijo, M. (1992) Immunology 75, 710. [31] Saito, S., Saito, M., Motoyoshi, K. and Ichij0, M. (1991) Biochem. Biophys. Res. Commun. 164, 284. [32] Nishino, E., Matsuzaki, N., Masuhiro, K., Kameda, T., Taniguchi, T., Takagi, T., Saji, F. and Tanizawa, O. (1990) J. Clin. Endocrinol. Metali. 71,436. [33] Akbar, A.N., Salmon, M. and Janossy, G. (1991) Immunol. Today 12, 184.
197
IMLET 02103
A study of CD45RO, CD45RA and CD29 antigen expression on human decidual T cells in an early stage of pregnancy •
a
*
S. Salto ' , K . N i s h i k a w a b, T. M o r i i b, N. N a r i t a b, M . E n o m o t o a, A. I t o a a n d M . Ichijo a aDepartment of Obstetrics and Gynecology, Nara Medical University, Nara 634, Japan; and bSeeond Department of Internal Medicine, Nara Medical University, Nara 634, Japan (Received 10 February 1992; accepted 10 November 1992)
1.
Summary
The decidua is the place where the fertilized egg is implanted and where the immunocompetent cells of the mother come into direct contact with genetically disparate cells of the conceptus. Although the T cells in the decidua are exposed to fetal antigens, the fetus is not rejected by maternal immunocompetent cells. In the present study, we examined surface markers to determine whether the T cells in the human decidua are naive T cells without or memory T cells with a history of antigen stimulation. Although few T cells were present in the decidua, as compared to the peripheral blood, CD45RO ÷, CD29 ÷ and C D 4 5 R A CD4 ÷ T cells as well as CD45RO ÷, CD29 ÷ and C D 4 5 R A - CD8 ÷ T cells, which are considered to be memory T cells, were in the majority, with only small numbers of C D 4 5 R O - , CD29and CD45RA ÷ CD4 ÷ and CD8 ÷ cells, which are naive T cells, present. Also, the decidual mononuclear cells secreted IL-2 and IL-4. Since IL-4 is secreted only by memory T cells, it is suggested that in the decidua memory T cells increase in number and secrete cytokines, thereby in some way influencing the phenomenon of fertility.
other using CD45RA, CD45RB and CD45RO antibodies [3-5]. T cells stained with CD45RO antibody are regarded as memory T cells (T cells which have a history of activation by antigens) [6-10]. The decidua is the place where the fertilized egg is implanted and the placenta grows. Recent studies have suggested that maternal T cells play an important role in maintaining pregnancy in mice [11-15]• In these reports, soluble T-lymphocyte factors (i.e., GM-CSF and IL3) enhance the proliferation of murine trophoblast cells in vitro. Wegrnan [12] proposed that immunostimulation, rather than suppression, is important in maintaining pregnancy. If this model is valid in humans, T cells in the decidua should possess the CD45RO and CD29 antigens because many cytokines are secreted from memory T cells. In this study, using flow cytometry, we examined whether the ratio of memory T cells is increased in the human decidua. Furthermore cytokine secretion (IL-2 and IL-4) from decidual mononuclear cells was examined. 3. Materials and Methods
3.1.
Isolation of mononuclear cells from decidual tissue
2. Introduction
CD45 gene expression is found in almost all cells of the hemopoietic system [1,2]. Because of differences in m R N A splicing, there are isoforms of the CD45 gene that can be distinguished from each Key words: CD45RO; Decidua; T cell; Cytokine; Human and memory T cell *Corresponding author: Shigeru Saito, Dept. of Obstetrics and Gynecology, Nara Medical University, 840 Shijo-cho, Kashihara-shi, Nara 634, Japan. SSDI 0 1 6 5 - 2 4 7 8 ( 9 3 ) 0 0 0 1 9 - N
Human decidual tissue samples from the early stages of pregnancy (weeks 6--11 of pregnancy) were cut into pieces and filtered through a nylon mesh (30 #1 in diameter). Then, by Ficoll-Hypaque gravity centrifugation, mononuclear cells were separated out. Prior to the study, informed consent was obtained from all subjects. 3.2.
Isolation of mononuclear cells from peripheral blood
Heparinized peripheral blood was subjected to Fi-
coll-Hypaque gravity m o n o n u c l e a r cells.
centrifugation
to
separate
4. Results
4.1. 3.3.
Flow cytometry
Phycoerythrin (PE)-labeled Leu-2 (CD8), PE-labeled Leu-3 (CD4) and fluorescein isothiocyanate (FITC)-labeled Leu-18 ( C D 4 5 R A ) were purchased from Becton Dickinson (USA). FITC-labeled U C H L - 1 ( C D 4 5 R O ) m o n o c l o n a l a n t i b o d y (mAb) was purchased f r o m Nichirei (Japan) and F I T C - l a beled 4B4 (CD29) was purchased f r o m Coulter (USA). Using a c o m b i n a t i o n o f PE-labeled and FITC-labeled mAbs, we conducted 2-color flow cytometry with an F A C S c a n (Becton Dickinson, USA).
3.4.
Determination o f lL-2 and IL-4
Decidual m o n o n u c l e a r cells were suspended in R P M I - 1 6 4 0 m e d i u m containing 10% fetal calf seru m at a concentration o f 1 x 106/ml, and 200 pl o f the suspension was placed into each well o f a 96-well microtiter plate. The supernatant was harvested 24 h later and stored at - 8 0 ° C . IL-2 was bioassayed with C T L L - 2 cells according to a previously reported m e t h o d [16]. The lowest detectable limit o f IL-2 in the samples o f the present study was 0.8 pM. IL-4 was assayed using an enzyme i m m u n o s o r b e n t assay kit (Amersham, UK). The lowest detectable limit o f IL-4 with this kit was 10 pg/ml.
3.5.
Expression of CD45RO, CD45RA and CD29 on CD4 + T-cell subset
O f peripheral blood lymphocytes (PBL) f r o m nonpregnant women, 41.1 ___8.1% (n = 10) were C D 4 +. O f C D 4 + cells, 44.0___7.4% (n = 1 0 ) showed C D 4 5 R O expression. O f P B L from 10 w o m e n in the early stages o f pregnancy, 36.9__ 6.8% were C D 4 + (Table 1). The percentage o f C D 4 ÷ cells a m o n g P B L did not differ between n o n - p r e g n a n t and pregnant women. O n the other hand, the percentage o f C D 4 5 R O ÷ cells a m o n g peripheral blood C D 4 + cells f r o m pregnant w o m e n was 5 5 . 5 _ 12.2%, which was significantly higher than the percentage for non-pregnant w o m e n ( P < 0 . 0 5 ) . O f decidual lymphocytes, 5 . 5 + 1.9% (n = 10) were C D 4 ÷. This percentage was a b o u t 1/7 o f the percentage for P B L from pregnant and n o n - p r e g n a n t women. The percentage o f C D 4 5 R O + cells a m o n g C D 4 + T cells from the decidua was 84.7___ 10.7%, which was significantly higher than the percentage for peripheral blood from n o n - p r e g n a n t and pregnant w o m e n ( P < 0 . 0 0 1 ) . We further analyzed the expression o f C D 4 5 R A and C D 2 9 on C D 4 + T-cell subset. As shown in Fig. 1, decidual C D 4 + cells bore high a m o u n t s o f C D 4 5 R O and C D 2 9 antigen, whereas decidual C D 4 + cells had low a m o u n t s o f C D 4 5 R A antigen. Identical findings were observed in two other individuals (data not shown).
Data analysis 4.2.
Values were expressed in mean__+SD. Student's t test was employed to test statistical significance.
Expression of CD45RO antigen on CD8 + T-cell subset
O f P B L from n o n - p r e g n a n t women, 24.2-t- 11.0% TABLE 1 CD45RO EXPRESSION ON DECIDUAL AND PERIPHERAL CD4 ÷ AND CD8 ÷ T CELLS All data indicate mean_ SD. CD4 + ( % ) a
CD45RO + CD4 + (%)a
CD45RO + CD4+/ CD4 + (%)b
CD8 + (%)a
CD45RO + CD8 + (%)a
CD45RO + CD8+/ CD8 + (%)¢
Non-pregnant women (PBL) n = 10
41.1 --- 8.1
17.8 + 2.8
44.0 - 7.4
24.2 + 4.8
6.3 ___2.2
27.1 + 11.0
Pregnant women (PBL) n = 10
36.9 __+6.8
20.2 + 5.0
55.5 + 12.2
24.8 + 5.9
8.5 + 3.9
34.3 + 14.3
5.5 ___ 1.9
4.5 _ 1.3
84.7 + 10.7
7.9 _ 5.6
4.3 + 2.5
58.4 ___ 13.2
Decidual lymphocyte n = 10
aData show the percentage of lymphocytes. bData show the percentage of CD45RO + CD4+/CD4 +. eData show the percentage of CD45RO + CD8+/CD8 +. 194
A. P B L (nonpregnant woman)
C. Decidual lymphocytes
B. P B L
(pregnant woman)
CD4
10 o
10 i
tO 2
. . . . . . .10 a
10°
IO'
i~12
1o
ta
l0 =
I0"
~ tO*
"';~ ' '"7,
IB
, 101
102
,,,,4 ........ l0 = 10'
CD 45 RO
o
A
. . . . . . . . . . . . . . . . . . . . . . i ...... t=l t(] 2 la ~ t
CD29
CD29
(3D 29
CD 45 RA
I0=
CD 45 RO
CD 45 RO
la I
CD4
CD 4
.... ';-",' "'T=,' '"';';~ ' '"';: CD 45 RA
............. ta I
I .............. 1o 2 1~ a
1
CD 45 RA
Fig. 1. CD45RO, CD29 and CD45RA expression on peripheral blood and decidual CD4 + T cells. PBL and decidual lymphocytes were stained with FITC-conjugated anti-CD45RO, CD29 and CD45RA and PE-conjugated anti-CD4. Sampleswere analyzed on a FACScan and displayed as histograms. The horizontal and vertical axes represent log fluorescence intensity and relative cell number, respectively.(upper column) CD45RO expression; (middle column) CD29 expression; and (lower column) CD45RA expression on CD4 ÷ cells. were CD8 + T cells. CD45RO was positive in 27.1 -t- 11.0% of CD8 + T cells. O f PBL from pregnant women, 2 4 . 8 + 5 . 9 % were CD8 + T cells and C D 4 5 R O was positive in 34.3___14.3% of CD8 + T cells (Table 1). The percentage of CD8 + T cells and the percentage of C D 4 5 R O + CD8 + T cells did not differ significantly between pregnant and non-pregnant women. On the other hand, the percentage of CD8 + cells among decidual lymphocytes was 7 . 9 + 5 . 6 % , which was about 1/4 of the percentage for PBL. The percentage of CD45RO ÷ cells among CD8 ÷ T cells from the decidua was 58.4+13.2%, which was significantly higher than the percentage for PBL from pregnant and non-pregnant women (P<0.001). In the decidua, most CD8 + T cells expressed CD29 antigens, whereas a few CD8 ÷ T cells expressed C D 4 5 R A antigens in all 3 cases (Fig. 2).
4.3.
IL-2 and IL-4 secretion of decidual mononuclear cells
IL-2 and IL-4 were identified in the supernatant of decidual mononuclear cells in 4 cases (Table 2). However, the levels of both cytokines in the supernatant of peripheral blood cells were below the detectable limit.
5. Discussion About 70-80% of h u m a n decidual lymphocytes are accounted for by C D 3 - C D 1 6 - CD56 bright natural killer cells called 'endometrial granulocytes' [1721]. The percentage of T cells in h u m a n decidual lymphocytes is lower than that in peripheral blood [1921]. Until now, very few reports on decidual T cells have been published. Recent reports suggested that maternal T cells play an important role in maintaining pregnancy in mice [11-15]. Bearing this in mind, we examined in the present study which phenotypes 195
A. P B L ( n o n p r e g n a n t woman)
B. PBL (pregnant woman)
CD 8
I0 °
I0'
CD8
I0= I0= 10"
I0 °
I0'
CD 45 RO
i~l I
15 3
I0 ~
10"
I~
11
% ~ , ,,,;~,, ,,,,;,;.,, ,,,,;;~,, ,,,;,, 4 CD 45 RA
CD 45 RA
I01
10 2
I0 ~
I0"
CD 45 RO
" , ' . ' "";,b' "",";~' "",'2~ ' '"';: CD29
.............. 1~] 2 '
10 2
CD8
CD 45 RO
" " " i ; ' " '"i;; -~'' '",';;~ .... CD29
~ ,,LI
C. Decidual lymphocytes
CD29
fll (
CD45 RA
Fig. 2. CD45RO, CD29 and CD45RA expression on peripheral blood and decidual CD8 + T cells. The horizontal and vertical axes represent log fluorescenceintensity and relative cell number, respectively.(upper column) CD45RO expression; (middle column) CD29 expression;and (lower column) CD45RA expression on CD8 + cells.
TABLE 2 IL-2 AND IL-4 PRODUCTION OF DECIDUAL MONONUCLEAR CELLS AND PERIPHERAL BLOOD MONONUCLEAR CELLSa Weeks of gestation
7 7 8 10
IL-2 (PM)
IL-4 (pg/ml)
decidua PBMCb
d e c i d u a PBMC
1.3 2.1 1.7 1.5
20 23 20 19
< 0.8 < 0.8 <0.8 <0.8
< 10 < 10 < 10 <10
aDecidual mononuclear cells and peripheral blood mononuclear cells were suspended in RPMI-1640 medium containing 10% fetal calf serum to a concentration of 1 x 106/ml. The cells were distributed in 0.2 ml aliquots into 96 round-bottomed wells and were cultured for 24 h. IL-2 concentration was determined by bioassay using IL-2-dependent CTLL-2 cells and IL-4 concentration was determined by EIA. bperipheral blood mononuclear cells. 196
o f T cells are present in the decidua (a place for imp l a n t a t i o n o f fertilized egg) primarily by analyzing the expression of C D 4 5 R O , C D 4 5 R A a n d CD29 antigen. I n this study, only a small n u m b e r of T cells was f o u n d in the decidua. However, most of the C D 4 + T cells a n d CD8 + T cells were m e m o r y cells which carried C D 4 5 R O a n d CD29 antigen a n d did n o t carry C D 4 5 R A antigen. C D 4 + m e m o r y T cells are reported to be a b u n d a n t in sites frequently exposed to recoil a n t i g e n (e.g., the skin, tracheal m u c o sa a n d intestinal mucosa) a n d in tissues affected by t u b e r c u l o u s pleurisy or arthritis [22-29]. I n these tissues, i m m u n o c o m p e t e n t cells are in an activated state. O u r previous study revealed that decidual T cells also express activation antigens such as CD69 a n d H L A - D R antigens [30]. It seems likely that the expression of C D 4 5 R O a n d CD29 antigens o n decidual T cells is the result of recognition of some fetal antigens by m a t e r n a l T cells. I n mice, M - C S F , G M C S F a n d IL-3 are reported to serve as placenta-proliferating factors [16]. Also in h u m a n s , M - C S F a n d IL-6 are reported to p r o m o t e the secretion a n d re-
lease o f h u m a n c h o r i o n i c g o n a d o t r o p i n f r o m the placenta [31,32]. C D 4 ÷ m e m o r y T cells are k n o w n to p r o d u c e large a m o u n t s o f c y t o k i n e s such as IL-2, IL-3, IL-4, IL-5, I L - 6 a n d I F N - ~ [33]. I n this study, we f o u n d t h a t dec i d u a l m o n o n u c l e a r cells secrete I L - 2 a n d IL-4. It has been r e p o r t e d t h a t IL-2 was p r o d u c e d by naive T cells a n d m e m o r y T cells [33]. H o w e v e r , I L - 4 was p r o d u c e d o n l y b y m e m o r y T cells [33]. These d a t a p r o v i d e evidence t h a t d e c i d u a l m e m o r y C D 4 ÷ T cells are i m m u n o c o m p e t e n t cells. It is highly p r o b able t h a t o t h e r c y t o k i n e s are p r o d u c e d by d e c i d u a l T cells a n d affect t r o p h o b l a s t g r o w t h o r differentiation. It was recently revealed t h a t C D 8 ÷ T cells also include m e m o r y T cells [9,10]. T h a t is, C D 4 5 R O ÷, C D 2 9 ÷ a n d C D 4 5 R A - C D 8 ÷ cells were recently f o u n d to possess a c y t o t o x i c activity. A l t h o u g h the significance o f an increase o f C D 8 ÷ m e m o r y cells in the d e c i d u a l tissue is u n k n o w n , this increase m a y be the result o f the sensitization o f the m a t e r n a l C D 8 T cells to some fetal cells. It is an o p e n q u e s t i o n w h e t h e r o t h e r c y t o k i n e s are p r o d u c e d b y d e c i d u a l C D 4 ÷ T cells a n d w h e t h e r C D 8 ÷ m e m o r y T cells exert a killer activity in the decidua.
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