1 Department of Physiology and 2 Department of Microbiology and Immunology, Dartmouth Medical School, Lebanon, NH 03756, USA
3 To whom correspondence should be addressed at: Department of Physiology, 708W Borwell, One Medical Center Drive, Lebanon, NH 03756, USA. Email: john.v.fahey{at}dartmouth.edu
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Abstract |
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Key words: chemokines/cytokines/female reproductive tract/innate immunity/polarized epithelial cells
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Introduction |
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Tight junctions between columnar epithelial cells maintain the integrity of the mucosal monolayer in the Fallopian tube, endometrium and endocervix. With an apical surface to the lumen and a basolateral surface to the basement membrane and underlying cells, epithelial cells have a structural, and often functional, polarized orientation. This permits epithelial cells to respond to different stimuli and serve as a directional conduit for different factors. For example, the epithelial cell pIgR transverses the epithelial cell from the basolateral side to the apical side to release IgA immunoglobins into the uterine lumen (Wira and Stern, 1992). Also, in intestinal epithelial cells, Toll-like receptor 5 (TLR5), the innate immune receptor for bacterial flagellin, is preferentially expressed on the basolateral side (Hershberg, 2002
). In other studies, uterine epithelial cells reportedly secrete cytokines such as transforming growth factor-
(TGF-
) preferentially at the basolateral surface and tumour necrosis factor-
(TNF-
) at the apical surface (Grant and Wira, 2003
).
Uterine epithelial cells in culture express TLRs that are capable of recognizing specific structural components of bacterial, fungal and viral pathogens (Schaefer et al., 2004; Young et al., 2004
). Addition of TLR agonists to epithelial cells often, but not always, elicits the induction and secretion of chemokines and cytokines, usually through intracellular signalling of NF-
B. The secreted pro-inflammatory mediators attract and stimulate the immune cells, often provoking further inflammation. Some epithelial secreted factors have multiple effects in innate and adaptive immunity. For example, the microbicide human
defensin-2, which we have shown is produced by primary polarized uterine epithelial cells in culture (Schaefer et al., 2005
), is also a chemokine for memory T cells and immature dendritic cells (Yang et al., 1999
) and neutrophils (Niyonsaba et al., 2004
). Similarly, the chemokine macrophage inflammatory peptide-3
(MIP-3
), which is secreted by uterine epithelial cells in response to lipopolysaccharide (LPS) (Crane-Godreau and Wira, 2004
) and attracts immature dendritic cells, has also been shown to be microbicidal for bacteria (Hoover et al., 2002
).
Whereas the induction and secretion of chemokines and cytokines from immune cells and FRT epithelial cells by TLR agonists have been demonstrated, the production, significance and interaction of these factors without microbial challenge has received less attention. Kaysili and associates reported on the importance of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in normal uterine physiology, particularly in proliferation, angiogenesis, menstruation, implantation, cervical ripening and parturition (Kayisli et al., 2002). The chemokines and cytokines regulate their own production as well as other chemokines/cytokines by autocrine and paracrine mechanisms (Haddad, 2002
). Also, sex hormones exert control over many chemokines/cytokines in the FRT (Wira et al., 2002
). For example, progesterone withdrawal results in upregulation of MCP-1 and IL-8, leading to chemotaxis and activation of monocytes and neutrophils, which results in release and activation of matrix metalloproteinases that contribute to initiation of menstruation (Critchley et al., 2001
). Thus, concentrations of chemokines and cytokines will vary in FRT tissues and fluids during normal physiological processes, as well as pathological conditions, such as infection and endometriosis.
Our objective in this study was to ascertain the constitutive production of several chemokines and cytokines by pure populations of FRT (Fallopian tube, endometrium and endocervix) epithelial cells grown in culture. By culturing the epithelial cells to high transepithelial resistance on cell inserts, we can mimic the in vivo conditions of compartmentalization between lumen and subepithelial tissues, and compare the cytokine/chemokine production with reported cytokine concentrations in FRT fluids, as well as with different FRT tissues. The presence of epithelial secreted chemokines and cytokines that are not microbially induced emphasizes the multiple effects that these factors have on normal FRT physiology, as well as the preparedness for potential pathogens. For example, constitutive chemokines and cytokines in luminal secretions of FRT tissues could provide a level of immune awareness to allow for immediate responsiveness to pathogenic microbes, both as microbicides and activators of immune cells should a breech of the epithelial barrier occur. In addition, several chemokines and cytokines have effects on proliferation and apoptosis that may contribute to maintenance of normal FRT architecture and environment. Also, epithelial factors may contribute to the quantity and type of immune cells trafficking in FRT tissues (Givan et al., 1997), the formation of uterine lymphoid aggregates (Yeaman et al., 1997
) and the sampling of luminal fluids for pathogenic microbials by leukocytes (Rescigno et al., 2001
). Finally, by assessing a number of chemokines and cytokines in each sample through Luminex analysis, we may find interesting patterns or networks of their production and regulation.
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Materials and methods |
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Epithelial cell sheets were separated from stromal cells by filtration through a 20 µm nylon mesh filter (Small Parts Inc., Miami Lakes, FL). Epithelial sheets were retained on the filters, while stromal cells passed through the filters. Washing and back-washing the filters with complete medium recovered the epithelial sheets. Epithelial sheets were collected, centrifuged at 500 g for 5 min and resuspended in a small volume of complete medium. Using this procedure, we have isolated epithelial cells, which stain positive for the epithelial antigens Ber-EP4 and cytokeratin and negative for CD4, CD45 and vimentin. Purity of epithelial cells was determined by flow cytometry. When stained with the pan-leukocyte marker CD45, non-epithelial cells were <0.1% of the total cells added to cell inserts (P.Wallace and C.Wira, unpublished data).
Cell culture
To establish a cell culture system of polarized human uterine epithelial cells with both apical and basolateral compartments, the human uterine epithelial cells were seeded into the apical compartment of human extracellular matrix- (BD Biosciences, Bedford, MA) coated Falcon cell culture inserts in 24-well culture dishes designed for these cell inserts (Fisher Scientific, Pittsburgh, PA). Typically, six, nine or 1224 cell inserts could be seeded with Fallopian tube, cervix or uterine tissue epithelial cells, respectively. For these experiments, apical and basolateral compartments had 300 and 600 µl of complete medium, respectively. The medium was changed every 2 days, and fungizone was removed from the complete medium 4 days after seeding. Maximum transepithelial resistance (TER) was achieved within 8 days for all tissues, and conditioned medium for assay was obtained after maximal TER was attained. Media from cell inserts with no cells were used as control blanks for the assays. Following each 48 h culture period, the apical and basolateral conditioned medium was removed, centrifuged at 10 000 g for 5 min in a microfuge (Eppendorf/Brinkman) to remove any cell debris and the supernatants were stored at 80 °C until assayed.
Measurement of transepithelial resistance
As an indicator of tight junction formation in epithelial cell monolayers, TER was assessed periodically using an EVOM electrode and Voltohmmeter (World Precision Instruments, Inc., Sarasota, FL), as described previously (Richardson et al., 1995). TER values were measured from a minimum of six inserts per patient and the means were calculated. These values were used to determine the mean TER for each tissue type as shown in Figure 1.
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Measurement of IL-8
Since concentrations of IL-8 in some samples exceeded the maximum on the IL-8 standard curve from the Luminex analysis, IL-8 in the apical and basolateral conditioned media from polarized epithelial cells was determined with an enzyme-linked immunosorbent assay (ELISA) Duoset test kit (R&D Systems). IL-8 standards were prepared in culture media. Calculations of IL-8 were determined from a standard curve after optical density measurements at 450 nm on an ELISA reader (Dynex). The level of detection for IL-8 was 15.6 pg/ml.
Statistics
The data for the uterine and Fallopian tube cells are presented as the mean ± SEM; cervical cell data are shown as the range. InSTAT Software was used to perform a one-way repeated-measures analysis of variance (ANOVA). When an ANOVA indicated that significant differences existed among means, pre-planned paired comparisons were made using the Dunnet method to adjust P-values. A P-value of <0.05 was considered statistically significant.
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Results |
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Cytokine and chemokine constitutive production by uterine epithelial cells
Figure 2 shows the apical and basolateral concentrations of IL-8, IL-6, G-CSF, MCP-1, GM-CSF, TNF and MIP-1
in 48 h conditioned media of polarized human uterine epithelial cells derived from six patients. IL-4 and IL-17 were below the level of detection in these analyses (not shown). For comparison with other studies that assessed FRT lavage fluids or cell culture media, data are shown as pg/ml; however, the basolateral medium in this investigation contains twice the volume of the apical medium (300600 µl). Therefore, this difference in volume must be considered when determining whether or not there is a preferential secretion of a particular cytokine/chemokine. The relatively small number of sample tissues does not permit a correlation between responses and menstrual status, patient age or reason for surgery.
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All cytokines/chemokines tested were consistently produced during weeks of culture, indicating that secretion is a constant aspect of the uterine epithelial cells. As shown in Figure 3, four successive 48 h conditioned apical media collections from uterine epithelial cells from one patient (2464) had comparable values of IL-6. Similar results were obtained with the other chemokines and cytokines, as well as from FRT epithelial cells derived from other patients (not shown).
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Discussion |
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Of the seven mediators secreted by the FRT epithelial cells, several are predominantly known for their chemotactic effect on leukocytes. For example, IL-8 and MCP-1 are potent chemokines for neutrophils (Baggiolini, 1995) and monocytes, (Yoshimura and Leonard, 1992
), respectively. Most pertinent is that we have demonstrated that the secretions from uterine epithelial cells in culture measured in this study have been shown to attract neutrophils (Shen et al., 2004
) and monocytes (R.Meter et al., unpublished data). The concentrations of IL-8 and MCP-1 in the FRT epithelial cell secretions were sufficient to achieve maximum effect in standard chemotaxis assays compared with recombinant chemokine. In addition, the chemotactic activity for neutrophils or monocytes was effectively removed by pre-incubation of the epithelial secretions with specific neutralizing antibodies to either IL-8 or MCP-1, respectively. GM-CSF, which is chemotactic for neutrophils (Gomez-Cambronero et al., 2003
), can greatly synergize the IL-8 effect; potentiation is best shown when GM-CSF is added at the same time as IL-8 (Shen et al., 2004
). MIP-1
is chemotactic for T cells, monocytes, neutrophils and natural killer (NK) cells (Menten et al., 2002
). Unlike IL-8 and MCP-1, the MIP-1
concentrations produced by the FRT epithelial cells in these studies are unlikely to have a strong direct chemotactic effect. The data presented in these studies underscore the critical role that epithelial cells have in controlling migration of leukocytes and lymphocytes in the FRT tissues.
In addition to the chemokines, the pro-inflammatory cytokines IL-6, G-CSF, GM-CSF and TNF- were constitutively produced by FRT epithelial cells. These mediators have significant effects on proliferation and differentiation of haematopoietic cells, as well as activation once the cells mature. IL-6 and TNF-
have multiple additional effects on immune cells, and these pleiotropic factors may even be considered as regulating inflammation when other pro-inflammatory factors are increasing without restraint.
In general, all FRT tissues tested produced a similar pattern of constitutive expression, with regard to both the type and relative quantity of chemokine and cytokine. The neutrophil chemokine IL-8 was secreted in the highest quantity by the epithelial cells of the three tissues. The values of 50 ng/ml for the FRT cells over 48 h are in the range of those found in normal and pathological cervical fluids (Simhan et al., 2003
). In addition to chemotaxis, high concentrations of IL-8 have been associated with proliferation and angiogenesis during early to mid-secretory phase, as well as with apoptosis during menstruation (Kayisli et al., 2002
), a process in which neutrophils contribute prominently. IL-6 was also produced in copious quantities by epithelial cells from all three tissues, with Fallopian tube cells producing approximately half the levels of the cervix and endometrium. Interestingly, G-CSF production was >6-fold higher from the uterine epithelial cells compared with the others. In addition, uterine GM-CSF and TNF-
were also produced in greater quantities relative to Fallopian tube and cervix. Since GM-CSF and TNF-
stimulate the production of G-CSF (Sieff et al., 1988
; Cavaillon, 1994
), it is possible that the former factors contribute to the latter's abundance by a positive feedback loop. Indeed, since production of cytokines and chemokines is often upregulated in an autocrine and/or paracrine manner, a simultaneous and/or sequential pattern of pro-inflammatory peptides may be produced by the FRT epithelial cells in order to marshal neutrophil, monocyte/macrophage, dendritic cell, T-cell and B-cell forces against a potential infective microbe. Chemokines are known to attract immune cells to tissues where cytokines affect differentiation and activation of immune cells. Vaday and colleagues have reported on the combination of inflammatory signals that can influence leukocyte actions, providing a variety of interactions (Vaday et al., 2001
). In addition to direct upregulation of their production, chemokines and cytokines can also modulate pro-inflammatory responses by increasing the number of chemokine and cytokine receptors on leukocytes. Finally, since there were no other cell types to respond to the plethora of chemokines and cytokines produced by the epithelial cells, feedback mechanisms and receptors on other cell types that might exist in vivo are not available in our culture system to attenuate (or even enhance) production.
All seven inflammatory mediators secreted by uterine epithelial cells showed a preferential secretion to the apical compartment of inserts, even after accounting for volume differences. Some factors, particularly IL-8 and IL-6, were also preferentially secreted apically by Fallopian tube and cervical epithelial cells; an apical tendency was observed with several others. Whether or not the relative lack of apical secretion by Fallopian tube epithelial cells is due to the relatively lower TER (see Figure 1) remains to be determined. Regardless, it is evident that epithelial cells from all three FRT sites produce chemokines and cytokines, and some of these secretions are preferentially released to the lumen. There are several potential benefits of this. These epithelial factors, which are modulated by sex hormones, undoubtedly contribute to the resident and temporary populations of immune cells in the subepithelial layers of the FRT tissues. For example, the chemokines produced by the epithelial cells could account for the influx of leukocytes and lymphocytes that form lymphoid aggregates observed during the secretory phase of normal endometrium (Yeaman et al., 1997). The immune cells, also under hormonal control, participate in architectural changes that occur during the menstrual cycle, as well as formation of the trophoblast in the presence of a conceptus. Should a breech in the epithelial lining occur, pre-formed chemokines and cytokines would fill the injured area and have immediate effects on immune cell trafficking and activation. Since some of the factors produced by epithelial cells [
-defensins, MIP, secretory leukocyte protease inhibitor (SLPI), etc.] are microbicidal, their luminal presence resists the development of microbial infection. In addition, luminal secretions of natural microbicides in the upper FRT tissues can most probably wash down to bathe the surface of the ectocervix and vagina to protect against potential pathogens. Finally, the relatively higher levels of chemokines and cytokines would attract macrophages and dendritic cells to the epithelial lining to sample the lumen for microbes. Thus, the preferential secretions contribute to innate immune surveillance.
The fact that the isolation of epithelial cell sheets results in epithelial cells that achieve high transepithelial resistance indicates that the cell population is essentially devoid of non-epithelial cells. In prior studies (Prabhala et al., 1998), we have shown by immunohistochemistry that the cells recovered are purified epithelial cells. This does not completely eliminate the possibility that a few inter-epithelial lymphocytes and/or leukocytes are present in our cultures. However, it is unlikely that a few non-epithelial cells could produce the concentration levels of cytokines and chemokines that we have found. In addition, we have shown that our epithelial cells do not secrete these chemokines and cytokines in response to LPS (T.M.Schaeffer et al., unpublished data). Since LPS routinely stimulates leukocytes to produce these pro-inflammatory factors, this is further evidence for the epithelial purity of our cultures.
The recruitment of specific populations of cells is essential for embryo implantation. For example, uterine NK cells increase in number rapidly during the secretory phase of the menstrual cycle and early pregnancy, and it is possible that the chemokines are responsible for these changes (Kitaya et al., 2003). The expression of chemokines that attract leukocytes is hormonally controlled, upregulated during the endometrial receptive period and associated with endometrial epithelia (Garcia-Velasco and Arici, 1999
; Caballero-Campo et al., 2002
; Dominguez et al., 2003
; Jones et al., 2004
). Our results showing constitutive production of several chemokines also suggest that epithelial cell production of chemokines could contribute to infiltration of NK cells and other leukocytes involved in altering endometrial architecture for embryo implantation.
Chemokines, cytokines, microbicides and other agents can act as autocrine and paracrine regulators in modulating human immunodeficiency virus (HIV). For example, chemokines, such as RANTES, MIP-1 and MCP that are produced by FRT epithelial cells, can act as CCR-5 antagonists to interfere with HIV uptake (Blanpain et al., 1999
). In a recent paper, we demonstrated increases in expression of the intracellular antiviral compounds interferon (IFN)-
, and the IFN-
-stimulated genes myxovirus resistance gene 1 and 2',5' oligoadenylate synthetase induced by poly(I:C) in pure populations of uterine epithelial cells (Schaefer et al., 2005
). In addition, we have shown that FRT epithelial cells constitutively produce SLPI (Fahey and Wira, 2002
), a known inhibitor of HIV infection (Pillay et al., 2001
). Studies are underway in our laboratory to elucidate the intracellular and secreted compounds from FRT epithelial cells with anti-HIV activity.
In conclusion, these studies suggest that FRT epithelial cells, through their secretions, contribute to maintaining physiological homeostasis, as well as preparedness in case of infectious insult. The constitutive secretion and compartmentalization of large quantities of bioactive chemokines and cytokines provide additional evidence for the role of epithelial cells as gatekeepers of innate immune protection in the female reproductive tract (Wira and Fahey, 2004). Further studies will explore the ways that sex hormones modulate both the constitutive and TLR-induced production and secretion of the epithelial chemokines and cytokines.
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Acknowledgements |
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Submitted on October 26, 2004; resubmitted on January 18, 2005; accepted on January 25, 2005.
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