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The mucosal epithelium is a major portal for microbial invasion. Mucosal barrier integrity is maintained by the physical interactions of intercellular junctional molecules on opposing epithelial cells. The epithelial mucosa in the female reproductive tract provides the first line of defense against sexually transmitted pathogenic bacteria and viruses, but little is known concerning the structure and molecular composition of epithelial junctions at this site. In the present study, the distribution of tight, adherens, and desmosomal junctions were imaged in the human endocervix (columnar epithelium) and ectocervix (stratified squamous epithelium) by electron microscopy, and permeability was assessed by tracking the penetration of fluorescent immunoglobulin G (IgG). To further define the molecular structure of the intercellular junctions, select junctional molecules were localized in the endocervical, ectocervical, and vaginal epithelium by fluorescent immunohistology. The columnar epithelial cells of the endocervix were joined by tight junctions that excluded apically applied fluorescent IgG. In contrast, the most apical layers of the ectocervical stratified squamous epithelium did not contain classical cell-cell adhesions and were permeable to IgG. The suprabasal and basal epithelial layers in ectocervical and vaginal tissue contained the most robust adhesions; molecules characteristic of exclusionary junctions were detected three to four cellular layers below the luminal surface and extended to the basement membrane. These data indicate that the uppermost epithelial layers of the ectocervix and vagina constitute a unique microenvironment; their lack of tight junctions and permeability to large-molecular-weight immunological mediators suggest that this region is an important battlefront in host defense against microbial pathogens.
Sexually transmitted infections (STIs) are epidemic worldwide and have far-reaching health, social, and economic consequences. Each year, more than 20 million men and women in the United States acquire an STI . The World Health Organization estimates the global annual incidence of curable STIs (excluding viral STIs) to be 333 million, of infections with human immunodeficiency virus type 1 (HIV-1) to be 3 million, and of herpes simplex virus type 2 to be 23.6 million . Some STIs, such as those involving HIV-1 and high-risk human papillomavirus strains, can cause severe morbidity, often leading to death. Others adversely affect fertility and neonatal health .
Epithelial surfaces in multicellular organisms constitute an interface that separates the individual from the environment. Epithelial intercellular junctions maintain the integrity and organization of epithelia by regulating molecular and cellular traffic and by providing a physical barrier to pathogen invasion. Three major types of cell-cell structural adhesions occur between epithelial cells: tight junctions, adherens junctions, and desmosomes [3, 4]. Tight junctions (zonula occludens) are composed of transmembrane proteins that make contact across the intercellular space and create a seal to restrict paracellular diffusion of molecules across the epithelial sheet [3, 5]. Tight junctions also have an organizing role in epithelial polarization by limiting the mobility of membrane-bound molecules between the apical and basolateral domains of the plasma membrane of each epithelial cell [3, 5]. Adherens junctions (zonula adherens) connect bundles of actin filaments from cell to cell to form a continuous adhesion belt, usually just below the tight junctions [4, 6]. Desmosomes (macula adherens) connect keratin intermediate filaments from cell to cell to form a structural framework of great tensile strength [4, 7].
Epithelial intracellular junctions contain distinctive combinations of specialized molecules. Tight junctions are comprised of a network of intermembrane fibrils of transmembrane proteins, including occludin, claudins, and junctional adhesion molecules (JAMs) . These proteins are linked to the cytoskeleton by cytosolic proteins such as the zona occludens proteins, which serve as adapter molecules and recruit regulatory proteins to the tight junction. The transcellular component of adherens junctions is comprised of epithelial cadherin (E-cadherin) dimers, anchored to the cytoskeleton via vinculin and alpha and beta catenin . The desmosomal adhesion proteins JAM3 (also known as [a.k.a.] JAM-C), desmoglein, and desmocollin are anchored to intermediate filaments via a scaffolding network of plakin and armadillo proteins .
Although once thought to be a rigid, static structure, the tight junction has a composition that can change rapidly in response to a range of stimuli, including estrogen, growth factors, calcium concentration, inflammatory mediators, and pathogen invasion [7–11]. Tight junctions are responsible for the sealing of the epithelial barrier as well as for the selective passage of small ions and fluid, which may be reliant on ion channels created by pore-forming claudins . As visualized by freeze-fracture electron microscopy, epithelial tight junctions contain four to nine protein strands; the number of strands directly correlates with the epithelial resistance of the tissues [13, 14].
A specialized mucosal epithelium covers internal surfaces. Much of the current knowledge of mucosal epithelial junctions is based on the gastrointestinal and pulmonary tracts [4, 5, 13]. Because the lower female genital tract is the primary site of infection by a number of sexually transmitted bacteria and viruses, an in-depth understanding of the structure and function of epithelial junctional complexes at this site is crucial to understanding immune defense and how STIs occur. However, only a few studies have begun to characterize intercellular junctions in human cervicovaginal tissue or cell lines [13–16]. We therefore undertook the present study to systematically characterize the structure, location, and molecular composition of epithelial intercellular junctions in normal human cervical and vaginal tissues. Our study focused on F11R (a.k.a. JAM-A), JAM3, occludin, E-cadherin, claudin-1, and TJP1 (a.k.a. ZO-1), which are junctional proteins thought to be key regulators of epithelial permeability, junctional integrity, and leukocyte infiltration [11, 17, 18]. After observing that the most apical layers in the stratified squamous epithelium of the ectocervix and vagina were devoid of tight junctions, we determined whether high-molecular-weight immunological mediators could infiltrate the apical layers of these tissues.
The present study was approved by the Institutional Review Boards of Boston University and Brigham and Women's Hospital in Boston, Massachusetts. Endocervical and ectocervical tissue specimens were obtained from discarded surgical samples from reproductive-aged (18–49 yr) and otherwise healthy patients undergoing hysterectomy. Discarded vaginal tissues were obtained from women undergoing vaginal repair procedures. Because the stratified squamous epithelium of the ectocervix is an extension of and continuous with the vagina , and because ectocervical tissues were more abundant, ectocervical tissue was utilized as the stratified squamous epithelial model for most of the present experiments.
Samples of endocervical and ectocervical tissues were minced into small cubes and fixed in 2.5% gluteraldehyde in cacodylate buffer (0.2 M sodium cacodylate, pH 7.6) at 5°C for 12 h. Tissues were then washed in cacodylate buffer and postfixed for 90 min in a 1:1 aqueous mixture of 2% osmium tetroxide and 3% potassium ferrocyanide. The fixed tissues were dehydrated through a graded series of ethanols and embedded in Spurr low-viscosity embedding medium. Ultrathin sections (thickness, 60–80 nm) were cut with an LKB Mark III ultramicrotome. Contrast was enhanced in ultrathin sections by sequential staining with a saturated uranyl acetate solution in 50% ethanol and 25% methanol for 10 min, followed by incubation in lead citrate. Ultrathin sections were examined with a Zeiss 10 electron microscope.
Endocervical and ectocervical samples were fixed and dehydrated to 100% ethanol as described above. These samples were then placed in the specimen chamber of a critical point drier (Polaron Institute). The dried samples were then mounted on scanning-electron microscope aluminum stubs with the mucosal surface facing up, coated with gold/palladium in a Hummer sputter coater (Technics), and viewed in an Autoscan microscope (Etec Systems) operated at 20 kV.
Endocervical (n = 5), ectocervical (n = 5), and vaginal (n = 3) epithelial tissues were fixed in 10% unbuffered, methanol-free formaldehyde. Tissues were then embedded in paraffin, and sections (thickness, 5–7 μm) were cut. Tissue sections were mounted on glass slides, dewaxed, and rehydrated in a graded series of alcohols. Before staining, sections underwent antigen retrieval. This was carried out by immersing the slides in a citrate buffer (pH 6; DAKO) in a decloaking chamber (Biocare Medical) that was brought up to 125°C for 30 sec. The slides were then allowed to cool, washed with distilled water, and placed in Tris buffer containing 0.1% Tween 20 (TBST; DAKO) for 5 min. Sections were blocked with 10% normal donkey serum for 30 min and quickly rinsed in TBST. The tissue sections were then treated with primary antibodies against one of the following epithelial adhesion molecules: human F11R (hJAM-A affinity purified goat immunoglobulin G [IgG]; R&D Systems), JAM3 (hJAM-C purified mouse IgG; R&D Systems), E-cadherin (mouse IgG1; Invitrogen), TJP1 (ZO-1 rabbit polyclonal; Santa Cruz Biotechnology), and claudin-1 (rabbit polyclonal; Zymed Laboratories), all at a dilution of 1:100 using a proprietary antibody diluent (DAKO) for 60 min. Tissues were then washed twice in TBST and incubated with either anti-mouse, anti-goat, or anti-rabbit IgG conjugated to Cy3 (Jackson ImmunoResearch Laboratories) at a dilution of 1:1000 in TBST for 30 min to visualize the primary antibody binding. All sections were mounted with Vectashield mounting medium (Vector Laboratories, Inc.) containing 4′,6′-diamidino-2-phenylindole (DAPI) as a nuclear counterstain and analyzed under an epiflourescence microscope (Olympus).
Endocervical and ectocervical specimens were processed within 45 min of surgical removal from the patient. The tissues were grossly dissected to remove extraneous connective tissue, leaving the epithelium and lamina propria intact, and then cut into pieces approximately 1 cm2 in size. Individual pieces of the explant tissues were placed mucosal side up in wells of a 12-well tissue culture plate (Costar Corning, Inc.) and cultured in 2 ml of keratinocyte serum-free medium (KSM; supplemented with bovine pituitary extract, epithelial growth factor, and CaCl2 plus penicillin/streptomycin; Jackson Labs) containing 100 μg/ml of preservative-free, Cy3-conjugated, whole-molecule human IgG (Jackson ImmunoResearch Laboratories). The tissues were then incubated at 37°C under 5% CO2 for 2 h. Samples were also incubated under the same conditions but in culture medium without labeled immunoglobulins to serve as negative controls.
Following incubation, tissue explant samples were washed vigorously in fresh medium twice for 5 min each time, fixed in 10% unbuffered methanol-free formaldehyde for 2 h, and processed for embedding in wax. Sections (thickness, 5 μm) were cut, dewaxed, hydrated, and then mounted in antifade mounting medium for fluorescent microscopy with DAPI as a fluorescing nuclear counterstain. Sections were examined under an Olympus BH2 microscope fitted with epifluorescence capabilities. Photos were taken in the middle of the tissue, away from the cut edges.
Scanning-electron microscopy of the endocervical luminal surface revealed a coherent, tightly packed layer of epithelial cells with abundant microvilli (Fig. 1A). In contrast, the luminal surface of ectocervical epithelia was composed of flattened, partially detached squamous epithelial cells (Fig. 1B).
When examined by transmission-electron microscopy, endocervical epithelial cells exhibited the classical tripartite junctional complexes typically associated with secretory mucosa. In the apical region, opposing endocervical cells were joined by juxtaluminal tight junctions, where fusion of adjacent cell membranes effectively seals the apex of each cell to prevent intercellular passage of luminal contents (Fig. 2, A and B). Below the tight junctions, adherens junctions were observed forming an adhesive band around adjacent cells, helping to maintain cohesion of the epithelium. Finally, desmosomes were observed below the adherens junctions, spaced at intervals around the cell circumference (Fig. 2, A and B).
In the ectocervical stratified squamous epithelium, exclusionary epithelial junctions were found beginning three to four cellular layers from the luminal surface, and abundant adherens junctions between neighboring cells were visualized in the suprabasal layers (Fig. 2, C and D). By contrast, the apical epithelial cells of the ectocervix, which are cornified and filled with glycogen, did not show any specialized adhesion junctions. Instead, they were loosely connected by short cellular projections that studded the surface of these cells (Fig. 2, E and F).
Immunofluorescence studies of the simple columnar epithelium of the endocervix revealed a similar distribution pattern for F11R and E-cadherin. Both were present at the contact between epithelial cells but not at the site of adhesion of epithelial cells to the basement membrane (Fig. 3, A and B). The distribution of claudin-1 was irregular; a variable expression pattern was observed associated with progenitor epithelial cells proximal to the basement membrane (Fig. 3C). JAM3 was not detected in the endocervical epithelium (Fig. 3D). TJP1 and occludin were localized between epithelial cells and also at the site of attachment to the basement membrane in some cells (Fig. 3, E and F). F11R, occludin, TJP1, and claudin-1 were also observed in the endothelium of blood vessels (Fig. 3, A and F, and data not shown), and TJP1 was expressed by fibroblasts in the lamina propria (Fig. 3E). Expression of E-cadherin and TJP1 was also visualized in the epithelial layer lining the endocervical glands (data not shown). Claudin-1 and occludin were also expressed, but to a lesser extent, along the basement membrane of endocervical glands (data not shown).
No junctional molecules were detected in the apical layers of the ectocervical or vaginal epithelia (Fig. 4, A–G). Tight junctional molecules were localized to the lower two thirds of the epithelium in a spiderweb-like pattern, indicative of their function in cell-cell adhesion (Fig. 4, A–G). E-cadherin and F11R had a similar pattern of distribution, with expression brightest in the parabasal and basal layers (Fig. 4, A and B). F11R expression appeared to be decreased in the rapidly proliferating epithelial cells directly adjacent to the basement membrane. This was also true of the junctional molecules claudin-1 and TJP1 (Fig. 4, C and E). Claudin-1 and JAM3 staining was most intense in the intermediate, parabasal, and basal epithelium and also around dermal papillae. Staining for TJP1 was less intense and localized to the parabasal and basal epithelium (Fig. 4E). TJP1 distribution was more punctate than the other junctional molecules studied and could also be visualized within the cytoplasm of the epithelial cells. This is in concordance with the known function of TJP1 as an intracellular scaffolding molecule. Expression of occludin was diffuse, and the intensity was highest in the parabasal epithelium (Fig. 4F). Because adhesion molecule expression patterns were identical in the ectocervical and vaginal epithelia, only images for the ectocervix are shown.
Cy3-labeled IgG was excluded by the tight junctions of the endocervical single-cell columnar epithelium (Fig. 5A). In contrast, fluorescent Cy3-labeled IgG penetrated the superficial layers of the ectocervical mucosal epithelium (Fig. 5B). As noted above, these apical layers are composed of cornified epithelial cells that do not appear to have exclusionary epithelial adhesion junctions. Below these cells, approximately three or four cellular layers from the lumen, epithelial junctions restricted the diffusion of labeled IgG between epithelial cells.
Cervicovaginal epithelial barrier integrity is maintained by intercellular junctions that prevent the invasion of microbes, with the exception of certain pathogenic organisms that have developed strategies to breech the epithelial barrier. Many other factors further fortify this barrier. Mucus produced by cervical and vaginal epithelial cells forms a glycocalyx on the epithelial surface that retains immunological mediators, including immunoglobulins and antimicrobial peptides [20–23]. Furthermore, a variety of leukocytes migrate into and through the epithelium to conduct immunosurveillance [23, 24]. The purpose of the present study was to characterize cervical and vaginal epithelial junctions to better understand their role in STI pathogenesis and immune defense of the lower female genital tract.
Our electron-microscopy studies indicate that classical tight junctions comprise the principal intracellular junctions between epithelial cells in the endocervix, in accordance with the current knowledge of the structure of simple columnar epithelia . These tight junctions formed a barrier that was impermeable to Cy3-labeled IgG. In contrast, the uppermost layers of the stratified squamous ectocervical epithelium were devoid of organized intracellular junctions, and the apical layers were permeable to Cy3-labeled IgG. Exclusionary junctions were observed directly beneath this layer, and IgG did not penetrate beyond this point.
The present study also surveyed the expression of discrete junctional molecules representing the different types of intracellular junctions in both the columnar epithelium of the endocervix and the stratified squamous epithelium of the ectocervix and vagina. The results from this investigation indicate that F11R, E-cadherin, occludin, claudin-1, and TJP1 are abundant junctional molecules in the human endocervix. JAM3, a desmosomal junctional molecule, was not detected in the endocervix. Claudin-1 expression was found in distinct foci, whereas the other junctional molecules were expressed uniformly at sites of cellular contact throughout the epithelium. Previous studies of cervical cancer biomarkers have described select junctional proteins in human cervical tissue. E-cadherin was previously described in the endocervical mucosa , and images published by Lee et al.  depicted irregular punctate claudin-1 staining localized to the basal cervical epithelial cells similar to that observed in the present study.
All of the intracellular junctional proteins surveyed in the present study, including JAM3, were detected in ectocervical and vaginal stratified squamous epithelia. The ectocervix is structurally a part of the vaginal wall and shares a continuous, morphologically identical mucosal layer with vaginal tissue . Therefore, it was not unexpected that the distribution of F11R, JAM3, claudin-1, TJP1, and E-cadherin was similar in these two tissues. Each of these molecules displayed a spiderweb-like distribution in the basal and suprabasal layers consistent with their functions as mediators of cell-cell adhesion. Little or no staining was observed in the most apical layers, where the epithelial cells gradually lose cell-cell contacts and are eventually sloughed into the lumen. Claudin-1, occludin, TJP1, and E-cadherin expression has been previously described in the human ectocervix [14, 27]. Claudin-4 and claudin-7 have been also detected in ectocervical and vaginal epithelial cells . To our knowledge, F11R expression has not been reported in cervical/vaginal epithelia, but it has been observed at other mucosal sites, such as the intestine and nasal epithelium [11, 17]. JAM3 expression has not been well studied in stratified squamous epithelia and has not been detected previously in the female mucosal epithelium, but it has been observed in the endothelium and retinal epithelium [18, 28]. The structure and distribution of adhesion molecules in the endocervical columnar epithelium and cervicovaginal squamous epithelium, as revealed by the present study, are diagrammed in Figure 6.
Some pathogens are known to affect the integrity of epithelial junctions to facilitate transmission across the mucosal surface. In the context of the female reproductive tract, Nazli et al.  showed that exposing female genital epithelial cells to free HIV virions or gp-120 envelope glycoprotein resulted in increased permeability. This correlated with increased production of the proinflammatory cytokine tumor necrosis factor-alpha and disruption of the tight junctional molecules TJP1, occludin, and claudin-1, -2, and -4 . Of interest, many junctional molecules also serve as leukocyte adhesion receptors, so they may play a role in the recruitment of CD4+ HIV target cells to the cervicovaginal lumen. F11R is a known ligand of lymphocyte-associated antigen 1 expressed on T cells, macrophages, and neutrophils; it may provide a foothold for migratory leukocytes [17, 29]. Similarly, E-cadherin is a receptor for the lymphocyte adhesion molecule alphaEbeta7 integrin on T cells .
In contrast to tight and adherens junctions, little evidence indicates that desmosomal structure is altered by pathogen invasion or inflammation . However, the desmosomal molecule JAM3 is a ligand for the macrophage-1 receptor on macrophages and neutrophils, and JAM3 regulates the influx of leukocytes, particularly neutrophils, in response to inflammatory stimuli . For a leukocyte to migrate between epithelial cells, the epithelial junctional bonds must be disrupted. Permeability to infiltrating leukocytes is largely regulated by secreted proinflammatory cytokines and chemokines .
A definitive understanding about the composition of cervical and vaginal epithelial junctions also provides an important foundation for future studies on pathogen transmission. For infections such as HIV, preventing epithelial barrier breach by cell-free or cell-associated virus is of the utmost importance . Results from the present study indicate that the uppermost layers of the stratified squamous epithelium covering the vagina and ectocervix may not comprise a physical barrier against STIs but, rather, a potential zone for interactions with immunological mediators that may be retained at this site. Mapping the normal expression of key molecular regulators of barrier resistance is an important first step in elucidating how microbes take advantage of these mechanisms to infect a host.
1Supported by National Institutes of Health grant R33 AI076966.