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Cell Biology International (2006) 30, 977982 (Printed in Great Britain)
Establishment of a mouse primary co-culture of endometrial epithelial cells and peripheral blood leukocytes: Effect on epithelial barrier function and leukocyte survival
Lok Sze Ho, Lai Ling Tsang, Yiu Wa Chung and Hsiao Chang Chan*
Epithelial Cell Biology Research Center, Department of Physiology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, N.T., HKSAR, Hong Kong
This study aimed to establish an in vitro co-culture model that would allow us to study the interaction between endometrial epithelial cells and immune cells. Flow cytometry analysis and cell surface marker staining were used to identify suitable immune leukocytes from a range of sources, such as intraepithelial lymphocytes (IEL), thymocytes, splenocytes and peripheral blood leukocytes. Optimizing culture conditions such as cell viabilities, cell seeding ratios and densities and co-culture methods were examined and determined. Results showed that co-culture of mouse endometrial epithelial cells (EEC) with peripheral blood leukocytes (PBL) at seeding densities of 3.0
Keywords: Co-culture, Endometrial epithelial cells (EEC), Immune cells, Peripheral blood leukocytes (PBL), Transepithelial resistance (TER).
*Corresponding author. Tel.: +852 2609 6839; fax: +852 2603 5022.
In addition to forming a barrier, epithelial cells, together with immune cells, are actively involved in the host defense against microbe infections. However, the interaction between epithelial and immune cells upon infections remains largely unknown.
Bacterial infections in the uterus can cause changes in epithelial function and stimulate a large number of immune cells to infiltrate the epithelium as defense mechanisms. Early studies have observed an increase in electrolyte secretion as measured by the short-circuit current (I
The paucity of information on interaction between epithelial cells and immune cells is largely due to the complexity of this interaction in vivo. Therefore, there is a great need for an in vitro co-culture model whereby interaction between specific types of cells can be assessed. The present study aimed to establish a primary mouse co-culture model of endometrial epithelial cells with various sources of immune cells. This was based on a primary mouse endometrial epithelial culture previously established in our laboratory, which has been used extensively to investigate epithelial transport mechanisms and regulation (Chan et al., 1997a,b,1999,2000a–c,2001,2002; Fong et al., 1998a,b; Tsang et al., 2001; Wang et al., 2001, 2002). A co-culture model with peripheral blood leukocytes exhibiting strong T-cell characteristics was eventually established based on its significant effects on leukocyte survival and epithelial barrier function.
2 Materials and methods
2.1 Materials, chemicals and antibodies
Dulbecco's modified Eagle's medium/Ham's F12 (DMEM-F12), antibiotics penicillin-streptomycin and trypsin (porcine pancreas) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA); while phosphate buffered saline (PBS), fetal bovine serum (FBS), non-essential amino acid (NEAA) and pancreatin were from Gibco Laboratories (Grand Island, NY, USA). Millipore filters were purchased from Millipore Corp. (Billerica, MA, USA). Matrigel basement membrane matrix was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Anesthetic ketamine (10%) and xylazine (2%) were from Alfasan International BV (Woerden, Holland). Anti-coagulating EDTA KE tubes were obtained from Sarstedt Ag. & Co. (Nürecht, Germany). Ficoll Paque-Plus and Percoll were purchased from Amersham Biosciences (GE Healthcare, NJ, USA). Mouse monoclonal anti-CD3+ FITC and anti-CD8+ PE antibodies were from BD Pharmingen (Franklin Lakes, NJ, USA), mouse monoclonal anti-cytokeratin 5 and 8 antibody was from Research & Diagnostic, Inc. (Flanders, NJ, USA) and mouse monoclonal anti-CD45+ PE antibody was purchased from Immunotech (Marselle Cedex 9, France).
2.2 Isolation of primary endometrial epithelial cells (EEC)
Endometrial epithelial cells were enzymatically isolated from mouse uteri according to the methods described (McCormack and Glasser, 1980), with slight modifications (Chan et al., 1997a). Briefly, uteri were obtained from 3-week-old immature ICR mice (body weight approximately 15
2.3 Isolation of peripheral blood leukocytes (PBL) and other immune cells
The concentration of anesthetic was prepared by mixing 0.75
Intraepithelial lymphocytes (IEL), residing in between epithelial cells, were isolated from mouse uteri by cutting the uteri longitudinally and enzymatically digesting the epithelium based on methods described for the isolation of IEL in rat gastrointestinal tracts (McKay et al., 1996; Kearsey and Stadnyk, 1996; Todd et al., 1999; Kerneis et al., 2000). IEL were further isolated using Percoll gradient centrifugation at 75% and 30% Percoll concentration diluted with 1× PBS and centrifuged at 400
2.4 Characterization of immune cells by flow cytometry
Immune cells of different sources including intraepithelial lymphocytes (IEL), thymocytes, splenocytes and peripheral blood leukocytes (PBL) were tested using flow cytometry (Beckman Coulter, USA). All isolated immune cells were stained with antibodies against CD3+ and CD8+ T-cell surface markers using anti-CD3+ FITC and anti-CD8+ PE antibodies (1:100) at RT for 1
2.5 Optimizing co-culture conditions
To establish the epithelial and immune cells co-culture, cell seeding ratios, cell viability and cell culture methods were examined. Cell seeding ratios were determined based on information suggested by references on rat vaginal histology and other gastrointestinal tract co-culture models (McKay et al., 1996; Kearsey and Stadnyk, 1996; Todd et al., 1999; Kerneis et al., 2000). Immune peripheral blood leukocyte viability was performed by trypan blue on cells right after isolation, after 4
2.6 Statistical analysis
Results were expressed as
3.1 Characterization of leukocytes by flow cytometry
Characterization of IEL by flow cytometry showed that only 5.9% of the IEL isolated exhibited both CD8+ and CD3+ T-cell characteristics in region E2 (Fig. 1a). Results of thymocytes showed high percentages of CD8+ and CD3+ T-cells (72.6%) in region E2, but most of these thymocytes were known to be immature (Fig. 1b). Flow cytometry results also showed that only very low numbers of splenocytes in region E2 (0.4%) exhibited CD8+ and CD3+ T-cell characteristics (Fig. 1c), while 67.5% of PBL in region E2 expressed both CD8+ and CD3+ characteristics, which were known to be mature T-cells (Fig. 1d). Based on the flow cytometry results, PBL were chosen for the subsequent co-culture experiments.
Flow cytometric analysis of immune cells with T-cell surface markers labeled with anti-CD3+ FITC and anti-CD8+ PE monoclonal antibodies. Regions E1 and E4 represent cells that either express CD8+ or CD3+ cell surface markers, E2 represents cells that express both CD8+ and CD3+ surface markers and E3 represents cells that express neither of the surface markers. A total of 10,000 cells were analyzed. (a) Intraepithelial lymphocytes (IEL); (b) thymocytes of the thymus; (c) splenocytes of the spleen and (d) peripheral blood leukocytes (PBL).
3.2 Cells seeding ratio and cell viability
Ratios of 1 immune cell (1.0
Immunofluorescent staining showing cell–cell contact between endometrial epithelial cell (EEC) and peripheral blood leukocyte (PBL) co-culture. Anti-cytokeratin FITC (green) monoclonal antibodies were used to label EEC and anti-CD45+ PE (red) monoclonal antibodies was used to label PBL.
Cell viability of PBL cells after isolation showed 95% cell survival as compared to after 4
Cell viability counts showing survival percentages of peripheral blood leukocytes after isolation (95%) compared to after co-culture with endometrial epithelial cells (EEC) for 4
3.3 Increased transepithelial resistance (TER) of EEC co-cultured with PBL
The effect of PBL on the EEC barrier function was examined by measuring the transepithelial resistance (TER) of the monolayers using short-circuit current (I
Measurement of transepithelial resistance (TER) in ‘mixed’ and ‘separate’ co-cultures of endometrial epithelial cells (EEC) and peripheral blood leukocytes (PBL). (a) TER measurement of ‘mixed’ co-culture showing increased TER in EEC/PBL co-culture (**p
To test whether the effect of PBL on EEC could be mediated by leukocyte-released factors such as cytokines, PBL were co-cultured with EEC but separated by a permeable membrane to allow free flow of cytokines but avoid cell–cell contact between the two types of cells. The results showed that there was no significant TER change in the co-culture (222.99
The present study explored the possibility of establishing an in vitro mouse co-culture model between endometrial epithelial cells (EEC) and immune cells of different sources. We made an effort to examine T-cell characteristics of the immune cells since they are thought to play a major role in local cellular immune response (Kelly et al., 2000; Robertson, 2000; Quayle, 2002; Johansson and Lycke, 2003; Kelly, 2003). The choice of IEL for the co-culture would have been ideal since they reside at the site of the endometrium, but it was experimentally impractical since only a very low number of IEL reside in the endometrial epithelium and to collect a significant amount of IEL for the co-culture would require an unreasonably large number of animals. Although the immune cells in the thymus and spleen were abundant, the thymus contains a large number of undeveloped T-cells and the spleen mainly possesses antibody-producing B-cells but not T-cells as shown by our flow cytometry results. The peripheral blood contains a large variety of circulating and maturing immune cells, with the majority of which exhibits T-cell characteristics shown by the flow cytometry results. During inflammation, recruitment of immune cells to the site of infection was also from the bloodstream. Therefore, the choice of PBL appeared to be suitable to mimic the endometrium environment for the future study of endometrial epithelial cell and immune cell interaction upon bacterial infections.
We based our current study on the ratios of immune cells to epithelial cells in the vagina of a rat, as reported by another study. The other study suggested that there was 1 immune cell per 100 epithelial cells (1:100) in the vagina of a rat under homeostasis, but this ratio quickly changes to 10 immune cells per 100 epithelial cells during bacterial infections (1:10) in the vagina (Sawicki et al., 1988). Based on this information, the EEC and PBL seeding ratio was determined to be 1:3 (PBL:EEC) during the initial cell culture, taking into account that EEC, but not PBL, will proliferate after 4
It is interesting to note that cell–cell contact plays a critical role in the interaction between endometrial epithelial cells and PBL since the effect of PBL on epithelial barrier function was diminished when they were co-cultured but separated by permeable support avoiding cell–cell contact. The effect of co-culture on leukocyte survival was also noted, which is likely to be mediated by cell–cell contact since we have previously observed that the co-culture effect on immune cell survival was greatly diminished when immune cells were co-cultured without cell–cell contact with intestinal epithelial cells (Xu et al., unpublished data). The present findings are consistent with the notion that cell–cell contact represents an important cross-talk mechanism between epithelial cells and immune cells, which is vital for immune cell survival and epithelial barrier function as demonstrated in the present study, as well as for cytokine release upon bacterial infection as demonstrated previously in the co-culture of intestinal epithelial cells and Peyer's patch lymphocytes challenged by Shigella lipopolysaccharide (LPS) (Chen et al., 2004).
In summary, the present study has established an in vitro co-culture model and demonstrated that endometrial epithelial-immune cell–cell contact is important in enhancing immune cell survival and epithelial protective barrier function. This co-culture model may enable future investigation of detailed cross-talk mechanisms between endometrial cells and immune cells upon a wide range of bacterial infections including Chlamydia trachomatis.
This work was supported by the Strategic Program of The Chinese University of Hong Kong.
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Received 25 November 2005/26 June 2006; accepted 19 July 2006doi:10.1016/j.cellbi.2006.07.004