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a Fearing Research Laboratory, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and tumor necrosis factor 
induced or significantly up-regulated expression of several of the cytokines and chemokines (i.e., IL-6, IL-8, RANTES, and M-CSF), as well as major histocompatibility complex (MHC) class II antigens, and membrane expression and shedding of the intercellular adhesion molecule-1 in all three cell lines. These data provide further evidence that epithelial cells in the lower human female genital tract participate in immunological functions, that their activity is up-regulated by proinflammatory/immune cytokines, and that epithelial cell immunological functions vary at different anatomical sites in the genital tract.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Most of the information on the role of epithelial cells in immune defense is derived from studies on columnar epithelial cell lines from human intestinal, respiratory, and urinary tracts [2–8]. Although mucosal epithelia at all anatomical sites share common barrier functions, female genital tract epithelial cells may have developed specialized characteristics to accommodate unique features of this mucosal site. The epithelium of the lower genital tract in women consists of two distinctive cell types. The epithelial lining of the vagina and ectocervix consists of multiple layers of stratified squamous epithelial cells; this is a nonsterile environment that tolerates an abundance of vaginal microorganisms as well as diverse seminal antigens. On the other hand, the endocervical epithelium consists of a single layer of columnar-type cells and forms a usually sterile passage into the upper genital tract. The endocervix also is the primary site of plasma and T cell localization in the lower genital tract [9]. Only a few studies to date have addressed cytokine expression by human cervical epithelial cells [10–13], and there have been no systematic comparative studies on the expression of immunological mediators by columnar epithelial cells of the endocervix versus stratified squamous epithelial cells from the ectocervix and vagina. We hypothesized that these two epithelial cell types produce distinct profiles of cytokines and other immunological mediators due to their different requirements for attraction and support of immune cell populations, tolerance of microorganisms, and maintenance of epithelial integrity. To test this hypothesis we studied three human papillomavirus (HPV)16/E6E7 immortalized epithelial cell lines from human vagina, ectocervix, and endocervix, established previously in our laboratory [14], for their ability to secrete various soluble immunological mediators under basal culture conditions and after stimulation with exogenous cytokines. Although immortalized, these cell lines maintain in vitro the phenotypic "fingerprints" of stratified squamous or simple epithelial cell types, respectively, as their cytokeratin and envelope protein profiles resemble those of epithelial cells in tissues of origin and "intact" primary cultures [14]. All three cell lines were derived through the same immortalization procedure; moreover, the endocervical and ectocervical cell lines were derived from the same individual, which minimizes differences in genetic background and, along with their unlimited potential for replication, makes them ideal for standardized cell type comparisons. We also tested the immortalized cell lines along with their parental primary cell lines and tissues of origin for the expression of immunologically relevant membrane molecules, including MHC class II molecules human leukocyte antigens-D-related (HLA-DR), the polymeric immunoglobulin receptor (pIgR), and the intercellular adhesion molecule-1 (ICAM-1).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The three HPV16/E6E7 immortalized epithelial cell lines, endocervical End1/E6E7, ectocervical Ect1/E6E7, and vaginal Vk2/E6E7, were maintained in keratinocyte serum-free medium (GIBCO BRL Life Technologies, Gaithersburg, MD) supplemented with 50 µg/ml bovine pituitary extract, 0.1 ng/ml epidermal growth factor, 100 U/ml penicillin, 100 µg/ml streptomycin, and CaCl2 to a final calcium concentration of 0.4 mM. The cell lines tested negative for mycoplasma by ELISA (Boehringer-Mannheim GmbH, Mannheim, Germany).
Detection of Soluble Immunobiological Mediators
The three cell lines were grown in Falcon 6-well culture plates (culture density 0.25 x 106 cells/ml) or T-25 culture flasks (culture density 1 x 106 cells/ml) (Becton Dickinson Labware; Becton Dickinson and Company, Franklin Lakes, NJ). Supernatants were collected at 30-min, 2-h, 6-h, 24-h, and 72-h intervals from confluent cell cultures. To minimize the effect of epithelial cell proliferation on dynamics of protein secretion, supernatants were collected at one single time point, with medium changed, respectively, at 30 min, 2 h, 6 h, 24 h, and 72 h prior to the supernatant collection. In addition, culture supernatants were collected from epithelial cells permeabilized with 0.5% Triton X-100 to assess intracellular (cytoplasmic) cytokine storage. Culture supernatants were immediately centrifuged for 10 min at 500 x g to remove cell debris and were stored in 0.5-ml aliquots at -80°C. For stimulation experiments, cultures were grown in Falcon 8-chamber slides (culture density 5 x 104 cells/ml) or 6-well plates (Becton Dickinson Labware). When the cultures reached confluence, tumor necrosis factor (TNF; 0.02 µg/ml), interferon (IFN; 0.1 µg/ml), interleukin (IL)-4 (0.025 µg/ml), or a mixture of IFN and IL-4 was added, and cultures were incubated for an additional 24 h. Human recombinant (r) TNF, rIL-4 and rIFN, purchased from R&D Systems (Minneapolis, MN), were used in these stimulation experiments.
Culture supernatants were assayed for cytokines and other immunologically relevant molecules by immunoenzyme kits purchased from R&D Systems and Genzyme (Cambridge, MA) (Table 1). Plates were read on an MRX Dynatech ELISA reader (Dynex Technologies, Chantilly, VA). The optical density readings and standard concentrations were plotted on a logarithmic scale, and optical densities were converted to picograms per milliliter using polynomial (quadratic) regression analysis (StatView II software; Abacus Concepts, Berkeley, CA). Results are presented as picograms or nanograms per 106 cells.
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Immunocytochemistry
Immunocytochemical analysis was performed on the immortalized cell lines to detect membrane expression of ICAM-1, MHC class II antigens, and pIgR. Primary cell lines (third passage) and frozen sections of normal ectocervical, endocervical, and vaginal tissues were used for comparison as in a previous study [14]. Cells were grown in 8-chamber slides for a minimum of 3 days before stimulation for 24 h with IFN (0.1 µg/ml) or TNF (0.02 µg/ml). Control (untreated) and stimulated cultures were washed and fixed with cold absolute methanol. An alkaline phosphatase biotin-streptavidin amplified system (StrAviGen; BioGenex, San Ramon, CA) and the Fast Red Substrate System K699 (Dako, Carpinteria, CA) were applied as described previously [14]. ICAM-1 was detected by Supersup anti-ICAM-1 (mixture of monoclonal antibodies R6.5, R6.1, CA7, CA8, specific for human CD54, courtesy of Boeringer Ingelheim Pharmaceuticals [Ridgefield, CT]), and the secretory component (SC) of the pIgR was detected by SC-05 monoclonal (Novocastra, Newcastle upon Tyne, UK). Since IFN and TNF induce MHC class II human leukocyte antigen D-related (HLA-DR) expression on most cell types including cervical cells [15], we used an anti-HLA-DR monoclonal TAL.1B5 from Dako as a positive control for epithelial cell activation by effector cytokines.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cytokines and chemokines
Constitutive release of cytokines and chemokines into culture supernatants was tested in parallel cultures of the three immortalized cell lines on at least five different occasions. The cytokine repertoire of the endocervical epithelial cells was highly reproducible and was qualitatively, as well as quantitatively, different from that of the ectocervical and vaginal cell lines. The endocervical epithelial cell line End1/E6E7 released substantial concentrations of 6 of 11 cytokines/chemokines tested in our experimental system, e.g. IL-6, IL-7, IL-8, macrophage colony-stimulating factor (M-CSF), transforming growth factor (TGF)ß1, and the chemokine known as "regulated-upon-activation, normal T cell expressed and secreted" (RANTES). In contrast, the ectocervical Ect1/E6E7 and the vaginal Vk2/E6E7 cell lines consistently released detectable levels of only 3 of 11 tested cytokines, i.e., IL-8, M-CSF, and TGFß1 (very low levels by Vk2/E6E7 cells). Trace amounts of macrophage inflammatory protein (MIP-1ß) were variably detectable in supernatants from all three cell lines; RANTES either was absent or was expressed in trace amounts by Ect1/E6E7 and Vk2/E6E7 cells; and MIP-1 was occasionally detectable at low concentrations in End1/E6E7 supernatants only.
Under basal (nonstimulated) conditions, as demonstrated in Figure 1, there were not only qualitative differences in cytokine/chemokine profiles, but also quantitative differences. The endocervical cell line not only produced more types of cytokines/chemokines, but in many cases also produced them in higher concentrations. Notably, IL-8 was detectable in End1/E6E7 culture supernatants as early as 30 min, and M-CSF and RANTES were detectable after 2 h, whereas they were detectable in Ect1/E6E7 and Vk2/E6E7 supernatants only after 6 h of culture. The other cytokines were detectable in 24- or 72-h supernatants only.
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Although IL-1ß and TNF were not spontaneously released into the culture media by any of the cell lines, both cytokines were detected at low concentrations after lysis of the cells with Triton X-100. In contrast, IL-6, IL-8, RANTES, MIP-1, and MIP-1ß were not detectable in the epithelial cell lysates, suggesting that the processing and secretion of these cytokines are concordant events. IFN was not detected in either cell lysates or supernatants from any of the cell lines.
IFN and TNF proved to be powerful effector cytokines for all three epithelial cell lines (Fig 2, A and B). IFN enhanced M-CSF release by all three lines (3–35 times), induced IL-6 release by the vaginal cells and up-regulated its release from the endocervical cells (1.9–4 times), and induced or up-regulated (1.5–27 times) RANTES release by all three cell lines. TNF was a more powerful and more consistent inducer of IL-8 release than IFN: TNF increased IL-8 production in all three cell lines (2.9–41 times), whereas IFN increased IL-8 secretion by the endocervical cell line only (3 of 6 cultures, 1.2–2.2 times). TNF was also a very strong stimulator of RANTES release by all three cell lines (9–12 times). In addition, it markedly up-regulated IL-6 release by the endocervical cells (up to 75 times) and stimulated its production by the ectocervical and vaginal epithelial cells. IL-4 was also tested as an effector cytokine on the cell lines, but had limited and inconsistent effects. It increased IL-8 in all three cell lines (1.3–9.3 times) and IL-6 in the endocervical cell line (3.06 times), but inhibited RANTES production by the endocervical cells. Interestingly, the combination of IL-4 and IFN decreased IL-8 production by all three cell lines (0.3–0.9 times). The following cytokines were not induced/enhanced by exogenous cytokine stimulation: TNF, IFN, IL-1ß, MIP1-, and MIP1-ß.
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Prostaglandin E2 (PGE2) and secretory leukoproteinase inhibitor (SLPI)
Abundant levels of PGE2 and SLPI were detectable in 24-h supernatants of all three epithelial cell lines, and concentrations were not affected by exposure of the cells to IFN or TNF. PGE2 reached plateau levels within 30 min, while SLPI increased approximately 6-fold over a period of 24 h. The vaginal cell line produced higher amounts of PGE2 than the ectocervical and endocervical cell lines (10.9 ± 2.4 vs. 1.7 ± 0.6 and 2.7 ± 0.7 ng/106 cells), whereas the ecto- and endocervical cell lines produced slightly higher levels of SLPI than did the vaginal cells (45.3 ± 11.3 and 47.3 ± 4.3 vs. 13.4 ± 1.7 ng/106 cells).
Expression and Shedding of MembraneReceptor Molecules
ICAM-1
Under basal conditions, minor cell subpopulations of both primary and immortalized epithelial cells from all three sites expressed cell-bound ICAM-1 (Fig. 3H); however, the soluble form of this adhesion molecule was not detectable in culture supernatants with the exception of low levels in vaginal cell supernatants. In vivo, membrane-bound ICAM-1 was rarely detectable in normal vaginal and cervical tissue. Scattered or clustered ICAM-1-positive epithelial cells were detected in 9 of 20 cases, predominantly in inflamed tissues (polymorphonuclear neutrophils [PMNs] visible) or the transformation zone (the squamocolumnar junction between the ecto- and endocervix) (Fig. 3G). Stimulation with TNF or IFN markedly increased the surface expression of ICAM-1 (Fig. 3, H and I) and caused abundant extracellular release of soluble (s) ICAM-1 by the three cell lines, with the ectocervical and vaginal cells producing more than the endocervical cells (Fig. 2, A and B). Expression of HLA-DR was up-regulated in all three cell lines by IFN and TNF (Fig. 3, K and L).
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Polymeric IgR In tissue sections, pIgR expression was markedly different among tissues originating from the three cervicovaginal regions. Abundant pIgR was detected in all endocervical specimens studied regardless of secretory or proliferative phase of the menstrual cycle, but expression was localized and rare in the ectocervical epithelium (specimens from 13 women examined) and absent in the vaginal epithelium (specimens from 7 women examined) (Fig. 3, A–D). Primary epithelial cells maintained these tissue-specific differences during their first passages (Fig. 3E). All immortalized lines, however, expressed pIgR without significant interlineage differences (Fig. 3F). Exogenous cytokines did not significantly change the amount of pIgR detectable by immunostaining.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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intraepithelial T cells that not only fight pathogens and directly kill infected cells, but also produce growth factors involved in healing epithelia damaged by infection or inflammation [21]. However, although IL-7 has been detected in cervicovaginal secretions, accumulations of T cells have not been observed in the lower female genital tract (J. Pudney, personal communication). TGFß, which was also detected in this study and has been documented in cervicovaginal secretions [32], is an important immunoregulatory cytokine, which also promotes wound healing [23] and localization of intraepithelial T cells by up-regulating the expression of CD103 (Eß7), enabling the T cells to adhere to E-cadherin expressed on mucosal epithelial cells [24].
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All three cervicovaginal cell lines secreted high levels of IL-8, M-CSF, PGE2, and SLPI. The constitutive presence of the proinflammatory cytokines IL-8 and M-CSF, which are also detectable at high concentrations in vivo in genital secretions [32], was balanced by the constant release of anti-inflammatory molecules, i.e. PGE2 and SLPI. Published reports have shown that PGE2 is produced constitutively as well as in response to inflammatory stimuli by cultured airway and gastrointestinal human epithelial cells [31, 33, 34]. It has also been detected in genital tract secretions [35]. The presence of SLPI in genital secretions and its localization to human genital tract epithelial cells in vivo have also been noted by a number of authors [36, 37]. This molecule is of interest because it suppresses HIV-1 infection [29]. Its presence in genital tract secretions may contribute to the relatively low sexual transmission rate of HIV-1 in healthy individuals.
All three cell lines expressed comparable levels of pIgR and ICAM-1. However, these antigens are more selectively expressed in vivo. In tissues, pIgR is primarily found in the endocervix, and ICAM-1 appears to be primarily expressed at sites of inflammation. Expression of these two molecules by both columnar and squamous epithelial cells in vitro suggests that both lineages are capable of expressing these molecules and that regional factors in situ may regulate their tissue expression.
Despite their different baseline cytokine profiles, the endocervical, ectocervical, and vaginal epithelial cells were all markedly responsive to effector cytokines such as TNF and IFN, which have been previously shown to modulate barrier functions of epithelial cells originating from other anatomical sites [38–40]. Our results showed that, similarly to what is seen in cultured intestinal epithelial cell lines [31, 41], ICAM-1 expression was dramatically up-regulated in cervical and vaginal epithelial cells in response to TNF and IFN. In addition, our study is the first to demonstrate that cytokine-activated epithelial cells can shed this molecule into the cervicovaginal environment. Shedding of soluble ICAM-1 has been noted for peripheral blood mononuclear cells, endothelial and melanoma cells, keratinocytes, and fibroblasts [30, 42]. Circulating ICAM-1 and other adhesion molecules have been reported to be elevated in human serum, cerebrospinal fluid, synovial fluids, and ascites in association with inflammation, infection, and cancers [30]. Experiments with recombinant soluble adhesins support speculations that they can block adhesion of leukocytes and thereby prevent extensive tissue damage; furthermore, they can bind to target lymphocyte function-associated antigen-1 (LFA-1)+ cells and deliver an activating signal without adhesion [30]. Thus, the release of soluble ICAM-1 by activated epithelial cells in the lower genital tract may have an important function in regulation of inflammation at this site.
In conclusion, our results indicate that epithelial cells in the human female genital tract play important and diverse roles in immune defense, and that epithelial cell differentiation pathways along with regulatory factors at different anatomical sites in the tissue environment affect these defense functions.
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FOOTNOTES |
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2 Correspondence: R.N. Fichorova, Fearing Research Laboratory, 75 Francis St., Thorn 2, Rm. 217, Boston, MA 02115. FAX: 617 264 5161; rfichorova{at}rics.bwh.harvard.edu
Accepted: September 25, 1998.
Received: July 9, 1998.
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