Molecular Human Reproduction Vol.8, No.2 pp. 176–180, 2002
Expression of interferon-γ-inducible protein-10 in human
endometrial stromal cells
Kengo Kai, Kaei Nasu1, Satomi Nakamura, Junichiro Fukuda, Masakazu Nishida and
Isao Miyakawa
Department of Obstetrics and Gynecology, Oita Medical University, Hasama-machi, Oita 879-5593, Japan
1To
whom correspondence should be addressed. E-mail: [email protected]
Human endometrial stromal cells (ESC) can produce a variety of chemokines, especially after inflammatory
stimulation. Interferon-γ-inducible protein-10 (IP-10) is a potent chemoattractant for lymphocytes, and belongs to
the family of non-ELR CXC chemokines. The expression of IP-10 in ESC after stimulation with interferon-γ
(IFN-γ), interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), or lipopolysaccharide (LPS) was evaluated using
an enzyme-linked immunosorbent assay and Northern blot analysis. A small amount of IP-10 protein was detected
in the culture media of unstimulated ESC. The expression of IP-10 mRNA was detected in ESC. IFN-γ, IL-1β,
TNF-α and LPS significantly stimulated the expression of IP-10 mRNA and protein in ESC. These results suggest
that the production of IP-10 by ESC is regulated by inflammatory mediators. The modulation of IP-10 concentrations
in the local environment may contribute to the normal and pathological processes of human reproduction by
regulating leukocyte trafficking in the endometrium.
Key words: endometrial stromal cell/interferon-γ-inducible protein-10/interferon-γ/interleukin-1β/tumor necrosis factor-α
Introduction
Human endometrial stromal cells (ESC) have been reported to
produce and secrete various chemokines, including interleukin
(IL)-8 (Arici et al., 1993; Nasu et al., 1998a,b, 1999a),
epithelial neutrophil-activating peptide-78 (ENA-78) (Nasu
et al., 2001a), growth-regulated oncogene (GRO)α (Nasu et al.,
2001b), monocyte chemoattractant protein-1 (MCP-1) (Nasu
et al., 1998a,b, 1999a), macrophage inflammatory protein
(MIP)-1α (Nasu et al., 1999a), and regulated upon activation,
normal T cell expressed and secreted (RANTES) (Arima et al.,
2000). The expression of these cytokines has been suggested
to be important in menstruation, bacterial infection, implantation, and in the maintenance of early pregnancy (Chard, 1995;
Garcia-Velasco and Arici, 1999).
Chemokines are a large superfamily of structurally and functionally related molecules with chemotactic activity targeted at
specific leukocyte populations. They are 70–90 amino acids in
length and are divided into four subfamilies based on the relative
position of their cysteine residues (CC, CXC, C, CXC3) (Miller
and Krangel, 1992; Baggiolini et al., 1994; Luster, 1998). The
CXC chemokines are further subdivided into two classes
depending on the presence of the glutamate–leucine–arginine
(ELR) motif preceding the first two cysteines (Baggiolini,
1998; Luster, 1998). IL-8, ENA-78, macrophage inflammatory
protein-2, GRO, and other members express this motif and
predominantly exert stimulatory and chemotactic activities
176
toward neutrophils (Baggiolini, 1998; Luster, 1998). In contrast,
interferon (IFN)-γ-inducible protein 10 (IP-10), monokine
induced by IFN-γ (MIG), and IFN-inducible T cell, a
chemoattractant (I-TAC) lack the ELR sequence and fail to
attract polymorphonuclear neutrophils (Baggiolini, 1998; Cole
et al., 1998; Luster, 1998). These non-ELR CXC chemokines
act on target cells through a CXCR3 motif (Loetscher et al.,
1996; Cole et al., 1998).
IP-10 is a 77 amino acid, 10 kDa protein that belongs to nonELR CXC chemokine family members (Luster et al., 1985;
Sarris et al., 1993). IP-10 shows pleiotrophic biological activities, including stimulation of monocytes, natural killer and T-cell
migration, regulation of T-cell and bone marrow progenitor
maturation, modulation of adhesion molecule expression, and
inhibition of angiogenesis (Neville et al., 1997). IP-10 expression is inducible by a variety of inflammatory mediators such as
IL-1, tumour necrosis factor (TNF)-α, IFN-α, IFN-γ and LPS in
monocytes (Luster et al., 1985; Cassatella et al., 1997), neutrophils (Cassatella et al., 1997), keratinocytes (Boorsma et al.,
1998), bronchial epithelial cells (Sauty et al., 1999), fibroblasts,
synovial cells (Luster et al., 1985; Bedard and Golds, 1993),
mesothelial cells (Visser et al., 1998) and endothelial cells
(Luster et al., 1985; Ebnet et al., 1996; Shields et al., 1999).
In addition, IP-10 expression within inflammatory lesions has
been implicated in lymphocyte recruitment to chronically
inflamed skin (Gottlieb et al., 1988), intestine (Uguccioni et al.,
© European Society of Human Reproduction and Embryology
IP-10 expression in endometrial stromal cells
Figure 1. Levels of interferon-γ-inducible protein-10 (IP-10) in the culture media of endometrial stromal cells after 24 h stimulation with
(A) interferon (IFN)-γ, (B) interleukin (IL)-1β, (C) tumour necrosis factor (TNF)-α, and (D) lipopolysaccharide (LPS). *P ⬍ 0.0025, **P ⬍
0.0001 versus unstimulated control (Bonferroni/Dunn test). The data are expressed as mean ⫾ SD of triplicate samples of the representative
of four separate experiments.
1999), kidney (Romagnani et al., 1999), liver (Shields et al.,
1999), and the central nervous system (Sorensen et al., 1999).
However, IP-10 expression in the human endometrium has not
been elucidated as yet.
In this study, we investigated the expression of the IP-10
transcript and protein by cultured ESC, which have been shown
to produce various CC and CXC chemokines, and we discuss
here the regulation of IP-10 expression in the cytokine network
in the endometrium.
Materials and methods
ESC isolation procedure
For the isolation and culture of ESC, nine endometrial specimens in the
late proliferative phase were utilized. The samples were obtained at
hysterectomy from patients with leiomyomas. Ethical approval of the
Institute was obtained, along with informed consent from all patients.
Normal ESC were separated from the epithelial glands by digesting the
tissue fragments with collagenase as previously described (Arici et al.,
1993; Nasu et al., 1998a,b). After three passages (15–20 days after
isolation) using standard methods of trypsinization, the cells were
⬎98% pure as analysed by immunocytochemical staining with
antibodies to vimentin (V9; Dako, Copenhagen, Denmark), keratin
(Dako), factor VIII (Dako), and leukocyte common antigen
(2B11⫹PD7/26, Dako) and, as such, were ready to be used for the
experiments.
Stimulation of ESC and detection of IP-10 in the culture media by
enzyme-linked immunosorbent assay (ELISA)
To study the production of IP-10 by ESC, 1⫻106 cells were plated on
6-well culture plates (Corning, New York, NY, USA) in 1 ml of Roswell
Park Memorial Institute 1640 medium (Gibco-BRL, Gaithersburg, MD,
USA) supplemented with 10% heat-inactivated FBS (Gibco-BRL) and
cultured until they were fully confluent. The supernatant was replaced
with fresh culture medium containing various amounts of recombinant
human IFN-γ (0.001–10 ng/ml) (R&D Systems, Minneapolis, MN,
USA), recombinant human IL-1β (0.001–10 ng/ml) (R&D systems),
recombinant human TNF-α (0.01–100 ng/ml) (R&D systems), and LPS
(Sigma, St Louis, MO, USA) (0.001–1 µg/ml). Under these conditions,
the supernatant was collected 24 h after stimulation and stored at –70°C
until assayed. The isolated cells from each patient were used for one
experiment at a time, and each experiment performed in triplicate
was repeated four times with cells from four different patients. The
concentrations of IP-10 were determined in the supernatants using a
commercially available ELISA (R&D systems). The sensitivity of the
assay for IP-10 was 15 pg/ml.
Detection of IP-10 mRNA in ESC by RT–PCR
To evaluate the expression of IP-10 mRNA in ESC, we amplified the
IP-10 transcript by means of the RT–PCR method using an RNA PCR
177
K.Kai et al.
Figure 2. Expression of mRNA for interferon-γ-inducible protein-10 (IP-10) and β-actin in endometrial stromal cells after 4 h stimulation
with various amounts of (A) interferon (IFN)-γ, (B) interleukin (IL)-1β, (C) tumour necrosis factor (TNF)-α, and (D) lipopolysaccharide
(LPS). Representative results of three separate experiments are shown.
kit with AMV RTase (Takara, Tokyo, Japan) as previously described
(Nasu et al., 1999b, 2001b). Total RNA was isolated from ESC
stimulated with IFN-γ (10 ng/ml) for 6 h using TRIzol reagent (GibcoBRL) according to the manufacturer’s instructions, and was reversetranscribed into complementary DNA (cDNA). To perform the PCR,
primer sets for IP-10 (sense primer: 5⬘-GTACCTCTCTCTAGAACCGTACG-3⬘; and antisense primer: 5⬘-GAGATCTTTTAGACATTTCC-3⬘) (Haicheur et al., 2000) were synthesized by the phosphoramide method on a DNA synthesizer (Model 8700; Biosearch, San
Rafael, CA, USA) and purified on Sephadex G50 columns (Pharmacia
LKB Biotechnology, Piscataway, NJ, USA) and by high-performance
liquid chromatography. The predicted size of the PCR product was 229
bp. The cDNA transcribed from 1 µg of total RNA was amplified using
a thermal cycler (Model PJ2000; Perkin Elmer, Norwalk, CT, USA).
The PCR with primer pairs for IP-10 was performed for 35 cycles, with
each cycle consisting of a denaturation step of 94°C for 1 min, an
annealing step of 55°C for 1 min, and an extension step of 72°C for
1 min. The PCR products were separated by 1.5% agarose gel (Takara)
electrophoresis and visualized by ethidium bromide (Takara) staining.
The PCR products were cloned using a TA cloning kit (Invitrogen,
Leek, The Netherlands) and used as the probe for Northern blot analysis.
Sequence analysis of the PCR products was also performed to confirm
that the amplified cDNA was IP-10.
Northern blot analysis for IP-10 mRNA expression in ESC
To study the expression of IP-10 mRNA in ESC, 5⫻106 cells were
plated on 75 cm2 culture flasks (Corning) in 15 ml of culture medium
with 10% heat-inactivated FBS and cultured until fully confluent.
The supernatant was replaced with fresh culture medium containing
various amounts of recombinant human IFN-γ (0.001–10 ng/ml),
recombinant human IL-1β (0.001–10 ng/ml), recombinant human
TNF-α (0.01–100 ng/ml), and LPS (0.001–1 µg/ml), and the cells were
further cultured for 4 h. Northern blotting was performed as previously
178
described (Nasu et al., 1999a,b, 2001b). Expression of β-actin mRNA
was also examined as an internal control. Each experiment was repeated
three times with cells from three different patients.
Statistical analysis
Data are presented as mean ⫾ SD and were appropriately analysed using
the Bonferroni/Dunn test employing StatView 4.5 (Abacus Concepts,
Berkeley, CA, USA). P ⬍ 0.05 was accepted as statistically significant.
Results
Detection of IP-10 protein in the culture media of ESC
The concentration of IP-10 in the culture medium without
cells was below the detection level. As shown in Figure 1,
low levels of IP-10 protein were detected in the culture medium
of non-stimulated ESC incubated for 24 h. The levels of IP10 were significantly increased with increasing concentrations
of IFN-γ, IL-1β, TNF-α and LPS.
IP-10 mRNA expression in ESC
IP-10 mRNA was detected by RT–PCR in IFN-γ-stimulated
ESC. The results of the sequence analysis of cDNA fragments
amplified by RT–PCR were consistent with the previously
reported sequence of human IP-10 (Luster et al., 1985). We used
this cDNA fragment as the probe for IP-10 in the Northern
blot analysis.
As shown in Figure 2, weak expression of IP-10 mRNA
was detectable in unstimulated ESC. Although there are some
deviations of the β-actin expression in some of the lanes, the
results show that IP-10 mRNA expression was significantly
induced by IFN-γ, IL-1β, TNF-α and LPS in a dose-dependent
manner.
IP-10 expression in endometrial stromal cells
Discussion
Chemokines are key components in the process of leukocyte
recruitment from vasculature into tissues. The interaction
of different chemokines with their receptors on leukocytes
allows for the selective activation and chemotaxis of neutrophils, eosinophils, lymphocytes or monocytes necessary for
migration to the sites of evolving inflammation. We have
previously reported the production of chemokines, IL-8 (Nasu
et al., 1998a,b, 1999a), ENA-78 (Nasu et al., 2001a), GROα
(Nasu et al., 2001b), MCP-1 (Nasu et al., 1998a,b, 1999a),
MIP-1 (Nasu et al., 1999a) and RANTES (Arima et al., 2000)
by human ESC and have suggested a paracrine regulation of
these chemokines in cyclic endometrium and during early
pregnancy.
IFN-inducible non-ELR CXC chemokines, such as MIG
and I-TAC, have not been demonstrated in the human endometrium. The present study first demonstrated the expression of
IP-10, a member of the non-ELR CXC chemokine family, in
ESC. IP-10 mRNA and protein were shown to be constitutively
expressed in cultured ESC and the expression of this molecule
was up-regulated by IFN-γ, IL-1β, TNF-α and LPS. We have
reported that the expression of IL-8, a member of the ELRcontaining CXC chemokine family, is enhanced by IL-1β,
TNF-α and LPS using the same experimental system (Nasu
et al., 1998a). In contrast, IFN-γ, a stimulator of IP-10
expression by ESC, inhibits the expression of IL-8 by ESC
(Nasu et al., 1998b). Since non-ELR CXC chemokines mainly
chemoattract and activate lymphocytes, whereas ELR-containing CXC chemokines act on neutrophils, the differential
regulation of these two subsets of CXC chemokines may
contribute to the trafficking of individual leukocyte subsets. It
is considered that inflammatory cytokines including IFN-γ, IL1β and TNF-α play important roles in the cytokine network
in human endometrium. These inflammatory cytokines may
also be implicated in the physiological control of IP-10
production by ESC, as well as in pathological conditions. The
modulation of IP-10 concentrations in the local environment
may contribute to the normal and pathological processes of
human reproduction by regulating lymphocyte trafficking in
the endometrium. Further investigations of non-ELR CXC
chemokines in endometrium may provide insight into the
mechanism involving the recruitment of inflammatory cells
during the human reproductive processes. Since the findings
of the present study are based on in-vitro experiments, further
studies may be necessary to elucidate the role of IP-10 in vivo.
Acknowledgments
This research was supported in part by the Ministry of Education,
Science, and Culture of Japan Grants-in-Aid 11770945 and 13770927
(to K.Nasu) and 13671733 (to I.Miyakawa) for Scientific Research.
References
Arici, A., Head, J.R., MacDonald, P.C. and Casey, M.L. (1993) Regulation of
interleukin-8 gene expression in human endometrial cells in culture. Mol.
Cell. Endocrinol., 94, 195–204.
Arima, K., Nasu, K., Narahara, H., Fujisawa, K., Matsui, N. and Miyakawa,
I. (2000) Effects of lipopolysaccharide and cytokines on production of
RANTES by cultured human endometrial stromal cells. Mol. Hum. Reprod.,
6, 246–251.
Baggiolini, M. (1998) Chemokines and leukocyte traffic. Nature, 392, 565–568.
Baggiolini, M., Dewald, B. and Moser, B. (1994) Interleukin-8 and related
chemotactic cytokines–CXC and CC chemokines. Adv. Immunol., 55,
97–179.
Bedard, P.A. and Golds, E.E. (1993) Cytokine-induced expression of mRNAs
for chemotactic factors in human synovial cells and fibroblasts. J. Cell.
Physiol., 154, 433–441.
Boorsma, D.M., Flier, J., Sampat, S., Ottevanger, C., de Haan, P., Hooft, L.,
Willemze, R., Tensen, C.P. and Stoof, T.J. (1998) Chemokine IP-10
expression in cultured human keratinocytes. Arch. Dermatol. Res., 290,
335–341.
Cassatella, M.A., Gasperini, S., Calzetti, F., Bertagnin, A., Luster, A.D. and
McDonald, P.P. (1997) Regulated production of the interferon-γ-inducible
protein-10 (IP-10) chemokine by human neutrophils. Eur. J. Immunol., 27,
111–115.
Chard, T. (1995) Cytokines in implantation. Hum. Reprod. Update, 1, 385–396.
Cole, K.E., Strick, C.A., Paradis, T.J., Ogborne, K.T., Loetscher, M., Gladue,
R.P., Lin, W., Boyd, J.G., Moser, B., Wood, D.E. et al. (1998) Interferoninducible T cell a chemoattractant (I-TAC): a novel non-ELR CXC
chemokine with potent activity on activated T cells through selective high
affinity binding to CXCR3. J. Exp. Med., 187, 2009–2021.
Ebnet, K., Simon, M.M. and Shaw, S. (1996) Regulation of chemokine gene
expression in human endothelial cells by proinflamatory cytokines and
Borrelia burgdorferi. Ann. NY Acad. Sci., 797, 107–117.
Garcia-Velasco, J.A. and Arici, A. (1999) Chemokines and human
reproduction. Fertil. Steril., 71, 983–993.
Gottlieb, A.B., Luster, A.D., Posnett, D.N. and Carter, D.M. (1988) Detection
of a gamma interferon-induced protein IP-10 in psoriatic plaques. J. Exp.
Med., 168, 941–948.
Haicheur, N., Escudier, B., Dorval, T., Negrier, S., De Mulder, P.H., Dupuy,
J.M., Novick, D., Guillot, T., Wolf, S., Pouillart, P. et al. (2000) Cytokines
and soluble cytokine receptor induction after IL-12 administration in cancer
patients. Clin. Exp. Immunol., 119, 28–37.
Loetscher, M., Gerber, B., Loetscher, P., Jones, S.A., Piali, L., Clark-Lewis,
I., Baggiolini, M. and Moser, B. (1996) Chemokine receptor specific for IP10
and mig: structure, function, and expression in activated T-lymphocytes. J.
Exp. Med., 184, 963–969.
Luster, A.D. (1998) Chemokines: chemotactic cytokines that mediate
inflammation. N. Engl. J. Med., 338, 436–445.
Luster, A.D., Unkeless, J.C. and Ravetch, J.V. (1985) γ-Interferon
transcriptionally regulates an early-response gene containing homology to
platelet proteins. Nature, 315, 672–676.
Miller, M.D. and Krangel, M.S. (1992) Biology and biochemistry of the
chemokines: a family of chemotactic and inflammatory cytokines. Crit.
Rev. Immunol., 12, 17–46.
Nasu, K., Matsui, N., Narahara, H., Tanaka, Y., Takai, N., Miyakawa, I. and
Higuchi, Y. (1998a) MaMi, a human endometrial stromal sarcoma cell
line that constitutively produces interleukin (IL)-6, IL-8, and monocyte
chemoattractant protein-1. Arch. Pathol. Lab. Med., 122, 836–841.
Nasu, K., Matsui, N., Narahara, H., Tanaka, Y. and Miyakawa, I. (1998b)
Effects of interferon-γ on cytokine production by endometrial stromal cells.
Hum. Reprod., 13, 2598–2601.
Nasu, K., Narahara, H., Matsui, N., Kawano, Y., Tanaka, Y. and Miyakawa,
I. (1999a) Platelet-activating factor stimulates cytokine production by
human endometrial stromal cells. Mol. Hum. Reprod., 5, 548–553.
Nasu, K., Sugano, T., Matsui, N., Narahara, H., Kawano, Y. and Miyakawa,
I. (1999b) Expression of hepatocyte growth factor in cultured human
endometrial stromal cells is induced through a protein kinase C-dependent
pathway. Biol. Reprod., 60, 1183–1187.
Nasu, K., Arima, K., Kai, K., Fujisawa, K., Nishida, M. and Miyakawa, I.
(2001a) Expression of epithelial neutrophil-activating peptide 78 in cultured
endometrial stromal cells. Mol. Hum. Reprod., 7, 453–458.
Nasu, K., Fujisawa, K., Arima, K., Kai, K., Sugano, T. and Miyakawa, I.
(2001b) Expression of growth-regulated oncogene α in human endometrial
stromal cells. Mol. Hum. Reprod., 7, 741–746.
Neville, L.F., Mathiak, G. and Bagasra, O. (1997) The immunobiology of
interferon-gamma inducible protein 10 kD (IP-10): a novel, pleiotropic
member of the C-X-C chemokine superfamily. Cytokine Growth Factor
Rev., 8, 207–219.
Romagnani, P., Beltrame, C., Annunziato, F., Lasagni, L., Luconi, M., Galli,
G., Cosmi, L., Maggi, E., Salvadori, M., Pupilli, C. et al. (1999) Role for
interactions between IP-10/Mig and CXCR3 in proliferative
glomerulonephritis. J. Am. Soc. Nephrol., 10, 2518–2526.
179
K.Kai et al.
Sarris, A.H., Broxmeyer, H.E., Wirthmueller, U., Karasavvas, N., Cooper, S.,
Lu, L., Krueger, J. and Ravetch, J.V. (1993) Human interferon-inducible
protein 10: expression and purification of recombinant protein demonstrate
inhibition of early human hematopoietic progenitors. J. Exp. Med., 178,
1127–1132.
Sauty, A., Dziejman, M., Taha, R.A., Iarossi, A.S., Neote, K., Garcia-Zepeda,
E.A., Hamid, Q. and Luster, A.D. (1999) The T cell-specific CXC
chemokines IP-10, Mig, and I-TAC are expressed by activated human
bronchial epithelial cells. J. Immunol., 162, 3549–3558.
Shields, P.L., Morland, C.M., Salmon, M., Qin, S., Hubscher, S.G. and Adams,
D.H. (1999) Chemokine and chemokine receptor interactions provide a
mechanism for selective T cell recruitment to specific liver compartments
within hepatitis C-infected liver. J. Immunol., 163, 6236–6243.
180
Sorensen, T.L., Tani, M., Jensen, J., Pierce, V., Lucchinetti, C., Folcik, V.A.,
Qin, S., Rottman, J., Sellebjerg, F., Strieter, R.M. et al. (1999) Expression
of specific chemokines and chemokine receptors in the central nervous
system of multiple sclerosis patients. J. Clin. Invest., 103, 807–815.
Uguccioni, M., Gionchetti, P., Robbiani, D.F., Rizzello, F., Peruzzo, S.,
Campieri, M. and Baggiolini, M. (1999) Increased expression of IP-10, IL8, MCP-1, and MCP-3 in ulcerative colitis. Am. J. Pathol., 155, 331–336.
Visser, C.E., Tekstra, J., Brouwer-Steenbergen, J.J.E., Tuk, C.W., Boorsma,
D.M., Sampat-Sardjoepersad, S.C., Meijer, S., Krediet, R.T. and Beelen,
R.H. (1998) Chemokines produced by mesothelial cells: huGRO-α, IP-10,
MCP-1 and RANTES. Clin. Exp. Immunol., 112, 270–275.
Submitted September 3, 2001; accepted November 26, 2001