Unidad de Inmunología, Instituto de Biotecnología, Universidad de Granada, E-18012 Granada, Spain
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words:
decidual stromal cell/desmin/myofibroblast/-smooth muscle actin/vimentin
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation and culture of decidual stromal cells
Samples of decidua from different patients were not mixed, to avoid alterations in the DSC phenotype resulting from allogeneic reaction and secretion of cytokines by lymphocytes that initially contaminate DSC cultures. Tissues were extensively washed in RPMI 1640 medium with 100 IU/ml penicillin and 50 µg/ml gentamicin, and the decidua was carefully freed from the trophoblast. It was then washed in Ca2+, Mg2+-free phosphate-buffered saline (PBS), minced, and left in a solution of 0.25% trypsin (Sigma, St Louis, MO, USA) and 0.02% EDTA (Merck, Darmstadt, Germany) for 15 min at 37°C. The enzymatic reaction was stopped by adding cold RPMI with 20% fetal calf serum (FCS) (Flow Laboratories, Irvine, UK); the suspension was filtered through gauze and centrifuged at 425 g for 7 min. The supernatant was discarded and the cell pellet was suspended in RPMI and centrifuged on Ficoll-Paque (Pharmacia Fine Chemicals, Uppsala, Sweden) for 20 min at 600 g. Cells were collected from the interface, suspended in RPMI and washed. This suspension, containing mainly DSC and leukocytes, was incubated in culture flasks for 1 h in complete RPMI with 10% FCS to allow macrophages and granulocytes to adhere to the flask. The supernatant was incubated overnight so that DSC adhered to the flask. Lymphocytes in the supernatant were then discarded, leaving a highly purified population of DSC free of granulocytes and macrophages. Proliferating DSC were expanded in culture for several passages; the medium was changed twice a week. In this work DSC maintained in culture were studied approximately 46 weeks after primary culture. During this period, proliferating DSC overgrew other possible contaminating cells. The purity of the culture was confirmed by the proportion of cells expressing both CD10 and CD13 and lacking CD14 and CD15 (Imai et al., 1992; Montes et al., 1996
). These cells express antigens associated with haematopoietic cells and have been previously identified as preDSC (Montes et al., 1996
; Olivares et al., 1997
).
Progesterone treatment
Decidual stromal cells isolated and maintained in culture as indicated above were cultured for 15 days in the presence of 100 nmol/l progesterone (preg-4-ene-3,20-dione, Sigma). This treatment led to decidualization, as shown by changes in cell morphology, expression of desmin, and expression or secretion of prolactin (Riddick and Kusmik, 1977; Glasser and Julian, 1986
; Tabanelli et al., 1992
; Olivares et al., 1997
).
Electron microscopy
Decidual stromal cells were fixed in 1% glutaraldehyde in 0.1 mol/l sodium cacodylate/0.1% sucrose buffer. Cells were washed in the same buffer and postfixed in 2% osmium tetroxide, dehydrated through a graded acetone series, and embedded in Epon 812. Thin sections were cut on a Reichert-Jung Ultracut E ultramicrotome, stained with lead citrate, and visualized and photographed in a transmission electron microscope (10C; Carl Zeiss, Inc., Oberkochen, Germany).
Monoclonal antibodies
The monoclonal antibodies (mAb) used in this study were anti-desmin (Eurodiagnostica, Apeldoorn, Holland); CAM 5.2 (cytokeratin) (Becton Dickinson, San Jose, CA, USA); OKM13 (CD13), OKM14 (CD14), OKM15 (CD15), anti-CD45 and OKDR (HLA-DR) (Ortho Diagnostic Systems, Beerse, Belgium); anti-human Thy-1, anti-vimentin (Serotec, Oxford, UK); BU38 (CD23) (The Binding Site; Birmingham, UK); anti--SM actin and anti-human follicular dendritic cells (HJ2) (Sigma); anti-human dendritic reticulum cells (DRC-1, CD21L) and anti-human common acute lymphoblastic leukaemia antigen (CD10) (DAKO, Glostrup, Denmark).
Flow cytometric analysis
Decidual stromal cells were washed and suspended in PBS at 106 cells/ml. Aliquots of 100 µl of the cell suspension were incubated with 10 µl of the appropriate mAb for 30 min at 4°C in the dark. Cells were washed, suspended in 1 ml PBS and immediately analysed in a flow cytometer (Ortho-Cytoron, Ortho Diagnostic Systems, Raritan, NJ, USA). As a negative control, cells were stained with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-labelled mouse IgG or IgM. The percentage of cells that were antibody-positive was calculated by comparison with the appropriate control. For double labelling, we followed the same procedure except that a second mAb with a different fluorescent marker from the first mAb was also added. For intracytoplasmic staining of cytoskeletal filaments, DSC were fixed with 4% paraformaldehyde for 20 min at 4 °C, and permeabilized with cold acetone for 10 min before the mAb was added.
Immunostaining
For immunostaining, cryostatic sections (5 µm) of early human decidua were fixed with acetone and labelled by an indirect immunoperoxidase method. Briefly, samples were rehydrated in PBS and incubated with hydrogen peroxide and AB human serum to block endogenous peroxidase and Fc receptors respectively. The samples were then incubated for 30 min at room temperature in a humid chamber with an appropriately diluted mAb. Normal mouse serum or an irrelevant mAb as negative controls were substituted for the first antibody. After three brief washings with PBS, samples were overlaid with peroxidase-conjugated goat antimouse IgG (Bio-Rad, Richmond, CA, USA) and diluted 1:100 in 1% PBS-BSA, and the reaction was developed with 0.5 mg/ml diaminobenzidine (Sigma) containing 0.01% hydrogen peroxide. The reaction was stopped after 510 min by washing in excess PBS. Samples were counterstained with Mayer's haematoxylin.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nevertheless, on the basis of the many immune activities reported for ESC and DSC (Tabibzadeh et al., 1989; Dudley et al., 1993
; Montes et al., 1995
; Olivares et al., 1997
; Ruiz et al., 1997
) and in view of their possible bone marrow origin (Lysiak and Lala, 1992
), some authors have proposed that DSC belong to the haematopoietic lineage (Lysiak and Lala, 1992
; Montes et al., 1996
). We previously demonstrated that human preDSC expressed antigens of myelomonocytic and B cell lineages, class II-HLA antigens, and low or undetectable levels of CD45 (Montes et al., 1996
), an antigenic profile that recalls that of the FDC (Schriever and Nadler, 1992
). The relationship between preDSC and FDC is further supported by the fact that preDSC express the FDC specific antigens DRC-1 and HJ2 (Figure 1
). Although FDC are dendritic cells with a clear immune function (antigen presentation to B lymphocytes in the lymphoid follicle), their spindle-shaped morphology, capacity for local self-renewal and failure to display the leukocyte common antigen (CD45) have led some authors to assign them to the mesenchymal rather than to the haematopoietic lineage (Schriever and Nadler, 1992
). The fact that preDSC also exhibited these latter mesenchymal features (Montes et al., 1996
; Olivares et al., 1997
), together with the expression of
-SM-actin by DSC (Figures 4 and 5
), a microfilament detected only in some cells of mesenchymal origin (Darby et al., 1990
), strongly support a mesenchymal lineage for these cells. Desmin, another cytoskeletal filament expressed by dDSC (Glasser and Julian, 1986
; Tabanelli et al., 1992
) (Figure 4
), is also found only in certain mesenchymal cells. The expression of vimetin (V) and
-SM-actin (A) (VA phenotype) by preDSC, and of vimetin,
-SM-actin and desmin (D) (VAD phenotype) by dDSC (Figure 4
) relates these cells to myofibroblasts (cells which exhibit the VA and VAD phenotypes), to vascular smooth muscle cells, and to pericytes (both of which show the VAD phenotype) (Schürch et al., 1992
). Furthermore, preDSC are located around the vessels (Ferenczy and Guralnick, 1983
), which is also where pericytes are typically found (Schürch et al., 1992
). Myofibroblasts, vascular smooth muscle cells and pericytes are probably related in their origin or differentiation, and they may even correspond to cellular isoforms (Schürch et al., 1992
). Thus, the cytoskeletal filament phenotype of DSC (Figure 4
) and morphological similarities between preDSC and myofibroblasts reported in this work (Figure 2
) strongly suggest that DSC are fibroblast-like cells that may also belong to the myofibroblast family. Recent observations have shown that FDC also express
-SM-actin (C.Oliver and E.G.Olivares, unpublished results), which indicates that these cells may also belong to this family.
The immunological activities and expression of haematopoietic antigens by the DSC do not contradict their possible mesenchymal origin, since it has been widely reported that fibroblasts and myofibroblasts express antigens associated with haematopoietic cells (Dongari-Bagtzoglou et al., 1997; Pechhold et al., 1997
; Sempowski et al., 1997
), secrete cytokines (Dongari-Bagtzoglou et al., 1997
; Sempowski et al., 1997
), costimulate T lymphocyte proliferation (Roberts et al., 1997
; Sempowski et al., 1997
) and also appear to be involved in transplant rejection (Pedagogos et al., 1997
). The possible bone marrow origin of the DSC (Lysiak and Lala, 1992
) is not incompatible with a mesenchymal origin, since stromal stem cells, which give rise to the progenitors of different mesenchymal cell types, were recently identified in bone marrow (Simmons and Torok-Storb, 1991
).
Members of the myofibroblast family exhibit contractile functions (for example, wound retraction for myofibroblasts or modulation of blood flow for pericytes) together with immune activities (Pedagogos et al., 1997; Roberts et al., 1997
). In the case of the DSC, although many immune functions have been observed in these cells (Tabibzadeh et al., 1989
; Dudley et al., 1993
; Montes et al., 1995
; Olivares et al., 1997
; Ruiz et al., 1997
); their contractile activity remains to be established. Nevertheless, the expression of
-SM-actin and the ultrastructural observation of abundant microfilaments in the cytoplasm of both pre and dDSC (Figures 15
) strongly suggest that these cells are contractile. The perivascular location of preDSC suggests that they may also play a role in blood flow regulation, while contractile DSC may collaborate in the expulsion of the trophoblast during abortion, or of the endometrium during menstruation. The immunological activities of DSC are predominantly associated with preDSC, whereas these activities are down-modulated in dDSC (Montes et al., 1995
; Ruiz et al., 1997
). The fact that a cytokine such as transforming growth factor-ß1 stimulates cell contractility (Moulin et al., 1998
) and blocks decidualization (Jikihara and Handwerger, 1994
; Kubota et al., 1997
) suggests that contractile activities may be carried out mainly by preDSC. Therefore, these cells may simultaneously perform contractile and immune functions in the elimination of trophoblast or endometrium. Nevertheless, further studies of the contractility of DSC will be needed to test this hypothesis.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Butch, A.W., Hug, B.A. and Nahm, M.H. (1995) Properties of human follicular dendritic cells purified with HJ2, a new monoclonal antibody. Cell. Immunol., 155, 2741.[ISI]
Darby, I., Skalli, O. and Gabbiani, G. (1990) -Smooth muscle actin is temporarily expressed by myofibroblasts during experimental wound healing. Lab. Invest., 63, 2129.[ISI][Medline]
Dongari-Bagtzoglou, A.I., Warren, W.D., Berton, M.T. et al. (1997) CD40 expression by gingival fibroblasts: correlation of phenotype with function. Int. Immunol, 9, 12331241.[Abstract]
Dudley, D.J., Trautman, M.S. and Mitchell, M.D. (1993) Inflammatory mediators regulate interleukin-8 production by cultured gestational tissues: evidence for a cytokine network at the choriodecidual interface. J. Clin. Endocrinol. Metab., 76, 404410.[Abstract]
Ferenczy, A. and Guralnick, M. (1983) Endometrial microstructure: structurefunction relationships throughout the menstrual cycle. Semin. Reprod. Endocrinol., 1, 205212.[ISI]
Fernández-Shaw, S., Shorter, S.C., Naish, C.E. et al. (1992) Isolation and purification of human endometrial stromal and glandular cells using immunomagnetic microspheres. Hum. Reprod., 7, 156161.[Abstract]
Foo, I.T., Naylor, I.L., Timmons, M.J. et al. (1992) Intracellular actin as a marker for myofibroblasts in vitro. Lab. Invest., 67, 727733.[ISI][Medline]
Franke, W.W. and Schinko, W. (1969) Nuclear shape in muscle cells. J. Cell. Biol., 42, 326331.
Glasser, S.R. and Julian, J.A. (1986) Intermediate filament protein as a marker for uterine stromal cell decidualization. Biol. Reprod., 35, 463474.[Abstract]
Imai, K., Maeda, M., Fujiwara, H. et al. (1992) Human endometrial stromal cells and decidual cells express cluster of differentiation (CD) 13 antigen/aminopeptidase N and CD10 antigen/neutral endopeptidase. Biol. Reprod., 46, 328334.[Abstract]
Jikihara, H. and Handwerger, S. (1994) Tumor necrosis factor- inhibits the synthesis and release of human decidual prolactin. Endocrinology, 134, 353357.[Abstract]
Kariya, M., Kanzaki, H., Takakura, K. et al. (1991) Interleukin-1 inhibits in vitro decidualization of human endometrial stromal cells. J. Clin. Endocrinol. Metab., 7, 11701174.
Kubota, T., Taguchi, M., Koboyashi, K. et al. (1997) Relationship between the release of prolactin and endothelin-1 in decidualized endometrial cells. Eur. J. Endocrinol., 137, 200204.[ISI][Medline]
Linge, C., Green, M.R. and Brooks, R.F. (1989) A method for removal of fibroblasts from human tissue culture systems. Exp. Cell. Res., 185, 519528.[ISI][Medline]
Loke, Y.W. and King, A. (1995) Human Implantation, Cell Biology and Immunology. University Press, Cambridge, pp. 102129.
Lysiak, J.J. and Lala, P.K. (1992) In situ localization and characterization of bone-marrow derived cells in the decidua of normal murine pregnancy. Biol. Reprod., 47, 603613.[Abstract]
Montes, M.J., Tortosa, C.G., Borja, C. et al. (1995) Constitutive secretion of interleukin-6 by human decidual stromal cells in culture. Regulatory effect of progesterone. Am. J. Reprod. Immunol., 34, 188194.[ISI][Medline]
Montes, M.J., Alemán, P., Tortosa, C.G. et al. (1996) Cultured human decidual stromal cells express antigens associated with hematopoietic cells. J. Reprod. Immunol., 30, 5366.[ISI][Medline]
Moulin, V., Castilloux, G., Auger, F.A. et al. (1998) Modulated response to cytokines of human wound healing myofibroblasts compared to dermal fibroblasts. Exp. Cell. Res., 238, 283293.[ISI][Medline]
Naiem, M., Gerdes, J., Abdulaziz, Z. et al. (1983) Production of a monoclonal antibody reactive with human dendritic reticulum cells and its use in the immunohistological analysis of lymphoid tissue. J. Clin. Pathol., 36, 167175.[Abstract]
Olivares, E.G., Montes, M.J., Oliver, C. et al. (1997) Cultured human decidual stromal cells express B71 (CD80) and B72 (CD86) and stimulate allogeneic T cells. Biol. Reprod., 57, 609615.[Abstract]
Oliveira, S.F., Nagata, T., Abrahamsohn, P.A. et al. (1991) Electron microscopic radioautographic study on the incorporation of 3H-proline by mouse decidual cells. Cell. Mol. Biol., 37, 315323.[ISI][Medline]
Pechhold, K., Patterson, N.B., Craighead, N. et al. (1997) Inflammatory cytokines IFN-gamma plus TNF-alpha induce regulated expression of CD80 (B71) but not CD86 (B72) on murine fibroblasts. J. Immunol., 158, 49214929.[Abstract]
Pedagogos, E., Hewitson, T.D., Walker, R.G. et al. (1997) Myofibroblast involvement in chronic transplant rejection. Transplantation, 64, 11921197.[ISI][Medline]
Richards, R.G. and Hartman, S. (1996) Human dermal fibroblast cells express prolactin in vitro. J. Invest. Dermatol., 106, 12501255.[Abstract]
Richards, R.G., Brar, A.K., Frank, G.R. et al. (1995) Fibroblast cells from term human decidua closely resemble endometrial stromal cells: Induction of prolactin and insulin-like growth factor binding protein-1 expression. Biol. Reprod., 52, 609615.[Abstract]
Riddick, D.H. and Kusmik, W.F. (1977) Decidua: a possible source of amniotic fluid prolactin. Am. J. Obstet. Gynecol., 127, 187190.[ISI][Medline]
Roberts, A.I., Nadler, S.C. and Ebert, E.C. (1997) Mesenchymal cells stimulate human intestinal intraepithelial lymphocytes. Gastroenterology, 113, 144150.[ISI][Medline]
Ruiz, C., Montes, M.J., Abadía-Molina, A.C. et al. (1997) Phagocytosis by fresh and cultured human decidual stromal cells. Opposite effects of interleukin-1 and progesterone. J. Reprod. Immunol., 33, 1526.[ISI][Medline]
Schriever, F. and Nadler, L.M. (1992) The central role of follicular dendritic cells in lymphoid tissues. Adv. Immunol., 51, 243284.[ISI][Medline]
Schürch, W., Seemayer, T.A., Gabbiani, G. (1992) Myofibroblast. In Sternberg, S.S. (ed.), Histology for Pathologists. Raven Press, New York, pp. 109144.
Sempowski, G.D., Chess, P.R. and Phipps, R.P. (1997) CD40 is a functional activation antigen and B7-independent T cell costimulatory molecule on normal human lung fibroblasts. J. Immunol., 158, 46704677.[Abstract]
Simmons, P.J. and Torok-Storb, B. (1991) Identification of stromal cell precursors in human bone-marrow by a novel monoclonal antibody, STRO-1. Blood, 78, 5562.[Abstract]
Tabanelli, S., Tang, B. and Gurpide, E. (1992) In vitro decidualization of human endometrial stromal cells. J. Steroid. Biochem. Mol. Biol., 42, 337344.[ISI][Medline]
Tabibzadeh, S.S., Santhanam, U., Sehgal, P.B. et al. (1989) Cytokine-induced production of IFN-beta-2/IL-6 by freshly explanted human endometrial stromal cells. Modulation by estradiol 17-. J. Immunol., 142, 31343139.
Submitted on September 15, 1998; accepted on February 10, 1999.