BIOLOGY OF REPRODUCTION 85, 97–104 (2011) Published online before print 6 April 2011. DOI 10.1095/biolreprod.110.090423

Structure and Function of Intercellular Junctions in Human Cervical and Vaginal Mucosal Epithelia1

Caitlin D. Blaskewicz,3 Jeffrey Pudney,4 and Deborah J. Anderson2,3,4,5

Departments of Molecular Medicine,3 Obstetrics and Gynecology,4 and Microbiology,5 Boston University School of Medicine, Boston, Massachusetts

ABSTRACT infections with human immunodeficiency virus type 1 (HIV-1) to be 3 million, and of herpes simplex virus type 2 to be 23.6 Downloaded from https://academic.oup.com/biolreprod/article/85/1/97/2530426 by guest on 27 September 2021 The mucosal is a major portal for microbial million [2]. Some STIs, such as those involving HIV-1 and invasion. Mucosal barrier integrity is maintained by the physical high-risk human papillomavirus strains, can cause severe interactions of intercellular junctional molecules on opposing morbidity, often leading to death. Others adversely affect epithelial cells. The epithelial mucosa in the female reproductive fertility and neonatal health [1]. tract provides the first line of defense against sexually transmitted pathogenic bacteria and viruses, but little is known Epithelial surfaces in multicellular constitute an concerning the structure and molecular composition of epithe- interface that separates the individual from the environment. lial junctions at this site. In the present study, the distribution of Epithelial intercellular junctions maintain the integrity and tight, adherens, and desmosomal junctions were imaged in the organization of epithelia by regulating molecular and cellular human endocervix (columnar epithelium) and ectocervix (strat- traffic and by providing a physical barrier to pathogen Downloaded from www.biolreprod.org. ified squamous epithelium) by electron microscopy, and invasion. Three major types of cell-cell structural adhesions permeability was assessed by tracking the penetration of occur between epithelial cells: tight junctions, adherens fluorescent immunoglobulin G (IgG). To further define the junctions, and [3, 4]. Tight junctions (zonula molecular structure of the intercellular junctions, select junc- occludens) are composed of transmembrane that make tional molecules were localized in the endocervical, ectocer- contact across the intercellular space and create a seal to restrict vical, and vaginal epithelium by fluorescent immunohistology. paracellular diffusion of molecules across the epithelial sheet The columnar epithelial cells of the endocervix were joined by [3, 5]. Tight junctions also have an organizing role in epithelial tight junctions that excluded apically applied fluorescent IgG. In polarization by limiting the mobility of membrane-bound contrast, the most apical layers of the ectocervical stratified molecules between the apical and basolateral domains of the squamous epithelium did not contain classical cell-cell adhe- plasma membrane of each epithelial cell [3, 5]. Adherens sions and were permeable to IgG. The suprabasal and basal junctions (zonula adherens) connect bundles of actin filaments epithelial layers in ectocervical and vaginal tissue contained the from cell to cell to form a continuous adhesion belt, usually just most robust adhesions; molecules characteristic of exclusionary below the tight junctions [4, 6]. Desmosomes (macula junctions were detected three to four cellular layers below the adherens) connect keratin intermediate filaments from cell to luminal surface and extended to the basement membrane. These data indicate that the uppermost epithelial layers of the cell to form a structural framework of great tensile strength [4, ectocervix and vagina constitute a unique microenvironment; 7]. their lack of tight junctions and permeability to large-molecular- Epithelial intracellular junctions contain distinctive combi- weight immunological mediators suggest that this region is an nations of specialized molecules. Tight junctions are comprised important battlefront in host defense against microbial patho- of a network of intermembrane fibrils of transmembrane gens. proteins, including , claudins, and junctional adhesion molecules (JAMs) [3]. These proteins are linked to the cervix, epithelium, junctions, permeability, vagina by cytosolic proteins such as the zona occludens proteins, which serve as adapter molecules and recruit INTRODUCTION regulatory proteins to the . The transcellular Sexually transmitted infections (STIs) are epidemic world- component of adherens junctions is comprised of epithelial wide and have far-reaching health, social, and economic (E-cadherin) dimers, anchored to the cytoskeleton via consequences. Each year, more than 20 million men and vinculin and alpha and beta catenin [5]. The desmosomal women in the United States acquire an STI [1]. The World adhesion proteins JAM3 (also known as [a.k.a.] JAM-C), Health Organization estimates the global annual incidence of , and desmocollin are anchored to intermediate curable STIs (excluding viral STIs) to be 333 million, of filaments via a scaffolding network of plakin and armadillo proteins [6]. 1Supported by National Institutes of Health grant R33 AI076966. Although once thought to be a rigid, static structure, the tight 2Correspondence: Deborah J. Anderson, Departments of Obstetrics/ junction has a composition that can change rapidly in response Gynecology and Microbiology, Boston University School of Medicine, to a range of stimuli, including estrogen, growth factors, calcium 670 Albany St., Suite 516, Boston, MA 02118. FAX: 617 414 8481; concentration, inflammatory mediators, and pathogen invasion e-mail: [email protected] [7–11]. Tight junctions are responsible for the sealing of the epithelial barrier as well as for the selective passage of small ions Received: 10 January 2011. and fluid, which may be reliant on ion channels created by pore- First decision: 8 February 2011. forming claudins [12]. As visualized by freeze-fracture electron Accepted: 21 March 2011. Ó 2011 by the Society for the Study of Reproduction, Inc. microscopy, epithelial tight junctions contain four to nine eISSN: 1529-7268 http://www.biolreprod.org strands; the number of strands directly correlates with the ISSN: 0006-3363 epithelial resistance of the tissues [13, 14]. 97 98 BLASKEWICZ ET AL.

A specialized mucosal epithelium covers internal surfaces. Sections were blocked with 10% normal donkey serum for 30 min and quickly Much of the current knowledge of mucosal epithelial junctions rinsed in TBST. The tissue sections were then treated with primary antibodies is based on the gastrointestinal and pulmonary tracts [4, 5, 13]. against one of the following epithelial adhesion molecules: human F11R (hJAM-A affinity purified goat immunoglobulin G [IgG]; R&D Systems), Because the lower female genital tract is the primary site of JAM3 (hJAM-C purified mouse IgG; R&D Systems), E-cadherin (mouse IgG1; infection by a number of sexually transmitted bacteria and Invitrogen), TJP1 (ZO-1 rabbit polyclonal; Santa Cruz Biotechnology), and viruses, an in-depth understanding of the structure and function claudin-1 (rabbit polyclonal; Zymed Laboratories), all at a dilution of 1:100 of epithelial junctional complexes at this site is crucial to using a proprietary antibody diluent (DAKO) for 60 min. Tissues were then understanding immune defense and how STIs occur. However, washed twice in TBST and incubated with either anti-mouse, anti-goat, or anti- only a few studies have begun to characterize intercellular rabbit IgG conjugated to Cy3 (Jackson ImmunoResearch Laboratories) at a dilution of 1:1000 in TBST for 30 min to visualize the primary antibody junctions in human cervicovaginal tissue or cell lines [13–16]. binding. All sections were mounted with Vectashield mounting medium We therefore undertook the present study to systematically (Vector Laboratories, Inc.) containing 40,60-diamidino-2-phenylindole (DAPI) characterize the structure, location, and molecular composition as a nuclear counterstain and analyzed under an epiflourescence microscope of epithelial intercellular junctions in normal human cervical (Olympus).

and vaginal tissues. Our study focused on F11R (a.k.a. JAM- Downloaded from https://academic.oup.com/biolreprod/article/85/1/97/2530426 by guest on 27 September 2021 A), JAM3, occludin, E-cadherin, claudin-1, and TJP1 (a.k.a. Tissue Permeability Studies ZO-1), which are junctional proteins thought to be key Endocervical and ectocervical specimens were processed within 45 min of regulators of epithelial permeability, junctional integrity, and surgical removal from the patient. The tissues were grossly dissected to remove leukocyte infiltration [11, 17, 18]. After observing that the extraneous connective tissue, leaving the epithelium and lamina propria intact, most apical layers in the stratified squamous epithelium of the and then cut into pieces approximately 1 cm2 in size. Individual pieces of the ectocervix and vagina were devoid of tight junctions, we explant tissues were placed mucosal side up in wells of a 12-well tissue culture determined whether high-molecular-weight immunological plate (Costar Corning, Inc.) and cultured in 2 ml of keratinocyte serum-free mediators could infiltrate the apical layers of these tissues. medium (KSM; supplemented with bovine pituitary extract, epithelial growth factor, and CaCl2 plus penicillin/streptomycin; Jackson Labs) containing 100

lg/ml of preservative-free, Cy3-conjugated, whole-molecule human IgG Downloaded from www.biolreprod.org. MATERIALS AND METHODS (Jackson ImmunoResearch Laboratories). The tissues were then incubated at

378C under 5% CO2 for 2 h. Samples were also incubated under the same Human Cervical and Vaginal Tissue conditions but in culture medium without labeled immunoglobulins to serve as negative controls. The present study was approved by the Institutional Review Boards of Following incubation, tissue explant samples were washed vigorously in Boston University and Brigham and Women’s Hospital in Boston, Massachu- setts. Endocervical and ectocervical tissue specimens were obtained from 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 discarded surgical samples from reproductive-aged (18–49 yr) and otherwise l healthy patients undergoing hysterectomy. Discarded vaginal tissues were (thickness, 5 m) were cut, dewaxed, hydrated, and then mounted in antifade obtained from women undergoing vaginal repair procedures. Because the mounting medium for fluorescent microscopy with DAPI as a fluorescing stratified squamous epithelium of the ectocervix is an extension of and nuclear counterstain. Sections were examined under an Olympus BH2 continuous with the vagina [19], and because ectocervical tissues were more microscope fitted with epifluorescence capabilities. Photos were taken in the abundant, ectocervical tissue was utilized as the stratified squamous epithelial middle of the tissue, away from the cut edges. model for most of the present experiments. RESULTS Transmission-Electron Microscopy Visualization of Cervical Epithelial Junctions Samples of endocervical and ectocervical tissues were minced into small by Electron Microscopy cubes and fixed in 2.5% gluteraldehyde in cacodylate buffer (0.2 M sodium cacodylate, pH 7.6) at 58C for 12 h. Tissues were then washed in cacodylate Scanning-electron microscopy of the endocervical luminal buffer and postfixed for 90 min in a 1:1 aqueous mixture of 2% osmium surface revealed a coherent, tightly packed layer of epithelial tetroxide and 3% potassium ferrocyanide. The fixed tissues were dehydrated cells with abundant microvilli (Fig. 1A). In contrast, the luminal through a graded series of ethanols and embedded in Spurr low-viscosity surface of ectocervical epithelia was composed of flattened, embedding medium. Ultrathin sections (thickness, 60–80 nm) were cut with an LKB Mark III ultramicrotome. Contrast was enhanced in ultrathin sections by partially detached squamous epithelial cells (Fig. 1B). sequential staining with a saturated uranyl acetate solution in 50% ethanol and When examined by transmission-electron microscopy, endo- 25% methanol for 10 min, followed by incubation in lead citrate. Ultrathin cervical epithelial cells exhibited the classical tripartite junctional sections were examined with a Zeiss 10 electron microscope. complexes typically associated with secretory mucosa. In the apical region, opposing endocervical cells were joined by Scanning-Electron Microscopy juxtaluminal tight junctions, where fusion of adjacent cell membranes effectively seals the apex of each cell to prevent Endocervical and ectocervical samples were fixed and dehydrated to 100% ethanol as described above. These samples were then placed in the specimen intercellular passage of luminal contents (Fig. 2, A and B). chamber of a critical point drier (Polaron Institute). The dried samples were Below the tight junctions, adherens junctions were observed then mounted on scanning-electron microscope aluminum stubs with the forming an adhesive band around adjacent cells, helping to mucosal surface facing up, coated with gold/palladium in a Hummer sputter maintain cohesion of the epithelium. Finally, desmosomes were coater (Technics), and viewed in an Autoscan microscope (Etec Systems) observed below the adherens junctions, spaced at intervals operated at 20 kV. around the cell circumference (Fig. 2, A and B). In the ectocervical stratified squamous epithelium, exclu- Immunofluorescence Assay sionary epithelial junctions were found beginning three to four Endocervical (n ¼ 5), ectocervical (n ¼ 5), and vaginal (n ¼ 3) epithelial cellular layers from the luminal surface, and abundant adherens tissues were fixed in 10% unbuffered, methanol-free formaldehyde. Tissues junctions between neighboring cells were visualized in the were then embedded in paraffin, and sections (thickness, 5–7 lm) were cut. suprabasal layers (Fig. 2, C and D). By contrast, the apical Tissue sections were mounted on glass slides, dewaxed, and rehydrated in a epithelial cells of the ectocervix, which are cornified and filled graded series of alcohols. Before staining, sections underwent antigen retrieval. with glycogen, did not show any specialized adhesion 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 1258C for 30 junctions. Instead, they were loosely connected by short sec. The slides were then allowed to cool, washed with distilled water, and cellular projections that studded the surface of these cells placed in Tris buffer containing 0.1% Tween 20 (TBST; DAKO) for 5 min. (Fig. 2, E and F). CERVICOVAGINAL EPITHELIAL JUNCTIONS 99 Downloaded from https://academic.oup.com/biolreprod/article/85/1/97/2530426 by guest on 27 September 2021

FIG. 1. Human endocervical and ectocervical luminal surfaces viewed by scanning-electron microscopy. A) The endocervical luminal surface showed an intact, cohesive epithelial cell layer. B) In contrast, the ectocervical luminal surface was covered with flattened and loosely attached epithelial cells. Downloaded from www.biolreprod.org. Original magnification 32000 (A) and 3200 (B).

Characterization of Junctional Molecules in Human molecules studied and could also be visualized within the Endocervical, Ectocervical, and Vaginal Epithelia cytoplasm of the epithelial cells. This is in concordance with the known function of TJP1 as an intracellular scaffolding by Immunofluorescence molecule. Expression of occludin was diffuse, and the intensity Endocervix. Immunofluorescence studies of the simple was highest in the parabasal epithelium (Fig. 4F). Because columnar epithelium of the endocervix revealed a similar adhesion molecule expression patterns were identical in the distribution pattern for F11R and E-cadherin. Both were ectocervical and vaginal epithelia, only images for the present at the contact between epithelial cells but not at the site ectocervix are shown. of adhesion of epithelial cells to the basement membrane (Fig. 3, A and B). The distribution of claudin-1 was irregular; a Permeability of Cervical Epithelia variable expression pattern was observed associated with progenitor epithelial cells proximal to the basement membrane Cy3-labeled IgG was excluded by the tight junctions of the (Fig. 3C). JAM3 was not detected in the endocervical endocervical single-cell columnar epithelium (Fig. 5A). In epithelium (Fig. 3D). TJP1 and occludin were localized contrast, fluorescent Cy3-labeled IgG penetrated the superficial between epithelial cells and also at the site of attachment to layers of the ectocervical mucosal epithelium (Fig. 5B). As the basement membrane in some cells (Fig. 3, E and F). F11R, noted above, these apical layers are composed of cornified occludin, TJP1, and claudin-1 were also observed in the epithelial cells that do not appear to have exclusionary endothelium of blood vessels (Fig. 3, A and F, and data not epithelial adhesion junctions. Below these cells, approximately shown), and TJP1 was expressed by fibroblasts in the lamina three or four cellular layers from the lumen, epithelial junctions propria (Fig. 3E). Expression of E-cadherin and TJP1 was also restricted the diffusion of labeled IgG between epithelial cells. visualized in the epithelial layer lining the endocervical glands (data not shown). Claudin-1 and occludin were also expressed, DISCUSSION but to a lesser extent, along the basement membrane of Cervicovaginal epithelial barrier integrity is maintained by endocervical glands (data not shown). intercellular junctions that prevent the invasion of microbes, Ectocervix and vagina. No junctional molecules were with the exception of certain pathogenic organisms that have detected in the apical layers of the ectocervical or vaginal developed strategies to breech the epithelial barrier. Many epithelia (Fig. 4, A–G). Tight junctional molecules were other factors further fortify this barrier. Mucus produced by localized to the lower two thirds of the epithelium in a cervical and vaginal epithelial cells forms a glycocalyx on the spiderweb-like pattern, indicative of their function in cell-cell epithelial surface that retains immunological mediators, adhesion (Fig. 4, A–G). E-cadherin and F11R had a similar including immunoglobulins and antimicrobial peptides [20– pattern of distribution, with expression brightest in the 23]. Furthermore, a variety of leukocytes migrate into and parabasal and basal layers (Fig. 4, A and B). F11R expression through the epithelium to conduct immunosurveillance [23, appeared to be decreased in the rapidly proliferating epithelial 24]. The purpose of the present study was to characterize cells directly adjacent to the basement membrane. This was cervical and vaginal epithelial junctions to better understand also true of the junctional molecules claudin-1 and TJP1 (Fig. their role in STI pathogenesis and immune defense of the lower 4, C and E). Claudin-1 and JAM3 staining was most intense in female genital tract. the intermediate, parabasal, and basal epithelium and also Our electron-microscopy studies indicate that classical tight around dermal papillae. Staining for TJP1 was less intense and junctions comprise the principal intracellular junctions between localized to the parabasal and basal epithelium (Fig. 4E). TJP1 epithelial cells in the endocervix, in accordance with the distribution was more punctate than the other junctional current knowledge of the structure of simple columnar epithelia 100 BLASKEWICZ ET AL.

FIG. 2. Visualization of epithelial junc- tions in the human endocervix and ecto- cervix by transmission-electron microscopy. A) Endocervix showing classic tripartite junctions (arrows) typically found in simple columnar epithelia. B) Higher magnification of endocervical adherens junctions. C)In the ectocervix, epithelial cells above the basement membrane are attached by robust adherens junctions (arrows). No discernable junctions were detected between epithelial (E) cells and an adjacent leukocyte (L). D) Higher-magnification image of C, with arrows indicating adherens junctions. E) Apical ectocervical surface, with arrows indicating no discernable junctions be- tween loosely associated epithelial cells. F) Downloaded from https://academic.oup.com/biolreprod/article/85/1/97/2530426 by guest on 27 September 2021 Higher-magnification image of E showing possible remnants of epithelial junctions (arrows). Original magnification 312 500 (A), 340 000 (B), 35000 (C), 325 000 (D and F), and 34900 (E). Downloaded from www.biolreprod.org.

[25]. These tight junctions formed a barrier that was are abundant junctional molecules in the human endocervix. impermeable to Cy3-labeled IgG. In contrast, the uppermost JAM3, a desmosomal junctional molecule, was not detected in layers of the stratified squamous ectocervical epithelium were the endocervix. Claudin-1 expression was found in distinct devoid of organized intracellular junctions, and the apical foci, whereas the other junctional molecules were expressed layers were permeable to Cy3-labeled IgG. Exclusionary uniformly at sites of cellular contact throughout the epithelium. junctions were observed directly beneath this layer, and IgG Previous studies of cervical cancer biomarkers have described did not penetrate beyond this point. select junctional proteins in human cervical tissue. E-cadherin The present study also surveyed the expression of discrete was previously described in the endocervical mucosa [26], and junctional molecules representing the different types of images published by Lee et al. [13] depicted irregular punctate intracellular junctions in both the columnar epithelium of the claudin-1 staining localized to the basal cervical epithelial cells endocervix and the stratified squamous epithelium of the similar to that observed in the present study. ectocervix and vagina. The results from this investigation All of the intracellular junctional proteins surveyed in the indicate that F11R, E-cadherin, occludin, claudin-1, and TJP1 present study, including JAM3, were detected in ectocervical CERVICOVAGINAL EPITHELIAL JUNCTIONS 101

FIG. 3. Junctional molecules of the endo- cervix, visualized by immunocytochemistry (Cy3-labeled antibodies appear red; DAPI- stained nuclei appear blue). All of the junctional molecules studied were detected in endocervical epithelial cell-cell junctions with the exception of JAM3, a desmosomal component. A) F11R (a.k.a. JAM-A). B)E- cadherin. C) Claudin-1. D) JAM3 (a.k.a. JAM-C). E) TJP1 (a.k.a. ZO-l). F) Occludin. G) Nonspecific mouse IgG control. H) Hematoxylin-and-eosin stain. Downloaded from https://academic.oup.com/biolreprod/article/85/1/97/2530426 by guest on 27 September 2021 Downloaded from www.biolreprod.org.

and vaginal stratified squamous epithelia. The ectocervix is but it has been observed at other mucosal sites, such as the structurally a part of the vaginal wall and shares a continuous, intestine and nasal epithelium [11, 17]. JAM3 expression has morphologically identical mucosal layer with vaginal tissue not been well studied in stratified squamous epithelia and has [19]. Therefore, it was not unexpected that the distribution of not been detected previously in the female mucosal epithelium, F11R, JAM3, claudin-1, TJP1, and E-cadherin was similar in but it has been observed in the endothelium and retinal these two tissues. Each of these molecules displayed a epithelium [18, 28]. The structure and distribution of adhesion spiderweb-like distribution in the basal and suprabasal layers molecules in the endocervical columnar epithelium and consistent with their functions as mediators of cell-cell cervicovaginal squamous epithelium, as revealed by the present adhesion. Little or no staining was observed in the most apical study, are diagrammed in Figure 6. layers, where the epithelial cells gradually lose cell-cell Some pathogens are known to affect the integrity of contacts and are eventually sloughed into the lumen. epithelial junctions to facilitate transmission across the mucosal Claudin-1, occludin, TJP1, and E-cadherin expression has surface. In the context of the female reproductive tract, Nazli et been previously described in the human ectocervix [14, 27]. al. [16] showed that exposing female genital epithelial cells to Claudin-4 and claudin-7 have been also detected in ectocer- free HIV virions or gp-120 envelope glycoprotein resulted in vical and vaginal epithelial cells [14]. To our knowledge, F11R increased permeability. This correlated with increased produc- expression has not been reported in cervical/vaginal epithelia, tion of the proinflammatory cytokine tumor necrosis factor- 102 BLASKEWICZ ET AL.

FIG. 4. Junctional molecules of the ecto- cervix and vagina visualized by immuno- cytochemistry (Cy3-labeled antibodies appear red; DAPI-stained nuclei appear blue). All junctional molecules studied were detected in both ectocervical and vaginal tissues, displaying a spiderweb-like pattern throughout the epithelium. Ectocervical staining patterns are shown here. A) F11R (a.k.a. JAM-A). B) E-cadherin. C) Claudin-1. D) JAM3 (a.k.a. JAM-C). E) TJP1 (a.k.a. ZO- l). F) Occludin. G) Nonspecific mouse IgG control. H) Hematoxylin-and-eosin stain. Downloaded from https://academic.oup.com/biolreprod/article/85/1/97/2530426 by guest on 27 September 2021 Downloaded from www.biolreprod.org.

alpha and disruption of the tight junctional molecules TJP1, cells, the epithelial junctional bonds must be disrupted. occludin, and claudin-1, -2, and -4 [16]. Of interest, many Permeability to infiltrating leukocytes is largely regulated by junctional molecules also serve as leukocyte adhesion secreted proinflammatory cytokines and chemokines [32]. receptors, so they may play a role in the recruitment of A definitive understanding about the composition of CD4þ HIV target cells to the cervicovaginal lumen. F11R is a cervical and vaginal epithelial junctions also provides an known ligand of lymphocyte-associated antigen 1 expressed on important foundation for future studies on pathogen transmis- T cells, macrophages, and neutrophils; it may provide a sion. For infections such as HIV, preventing epithelial barrier foothold for migratory leukocytes [17, 29]. Similarly, E- breach by cell-free or cell-associated virus is of the utmost cadherin is a receptor for the lymphocyte adhesion molecule importance [33]. Results from the present study indicate that alphaEbeta7 integrin on T cells [30]. the uppermost layers of the stratified squamous epithelium In contrast to tight and adherens junctions, little evidence covering the vagina and ectocervix may not comprise a indicates that desmosomal structure is altered by pathogen physical barrier against STIs but, rather, a potential zone for invasion or inflammation [31]. However, the desmosomal interactions with immunological mediators that may be molecule JAM3 is a ligand for the macrophage-1 receptor on retained at this site. Mapping the normal expression of key macrophages and neutrophils, and JAM3 regulates the influx of molecular regulators of barrier resistance is an important first leukocytes, particularly neutrophils, in response to inflamma- step in elucidating how microbes take advantage of these tory stimuli [18]. For a leukocyte to migrate between epithelial mechanisms to infect a host. CERVICOVAGINAL EPITHELIAL JUNCTIONS 103 Downloaded from https://academic.oup.com/biolreprod/article/85/1/97/2530426 by guest on 27 September 2021

FIG. 5. Cervical permeability to Cy3-labeled (red) IgG. The endocervical epithelium provided a robust barrier to penetration of Cy3-tagged IgG, whereas the most apical layers of the ectocervix were permeable. A) Endocervical tissue incubated with Cy3-labeled IgG. No diffusion is seen into the epithelium Downloaded from www.biolreprod.org. past the tight junctional complex (left arrow). The arrow on the right indicates a compromised epithelial cell. B) Cy3-labeled IgG penetrated the apical cellular layers of ectocervical epithelium. Original magnification 3750 (A) and 3600 (B).

FIG. 6. Localization of selected interepithelial adhesion molecules in the female lower genital tract. A) The endocervical epithelium contains classic tripartite junctions. The tight junctions are located near the apical surface; they seal the epithelium and maintain cellular polarity. Adherens junctions are located directly below the tight junctions and are primarily responsible for cell-. E-cadherin is the common transcellular component of all epithelial adherens junctions and is anchored to the actin cytoskeleton by vinculin and alpha and beta catenins. Desmosomes are the most basal adhesion structure and endow the tissue with mechanical resistance and strength. The major extracellular components of the are desmoglein and desmocollin, which connect to intermediate filaments of adjacent cells through an intracellular scaffolding network. B) The most robust junctions in the stratified squamous epithelium of the ectocervix and vagina lie in the parabasal epithelium, just above the basal layer in contact with the basement membrane. Adherens junctions are particularly abundant. The integrity of the junctions progressively lessens as epithelial cells are pushed toward the apical surface, where they become cornified, lose all cellular contacts, and are sloughed into the lumen. 104 BLASKEWICZ ET AL.

REFERENCES a component of desmosomes and a ligand for CD11b/CD18-mediated neutrophil transepithelial migration. Mol Biol Cell 2004; 15:3926– 1. Centers for Disease Control and Prevention. Sexually Transmitted Disease 3937. Surveillance 2009. Atlanta, GA: U.S. Department of Health and Human 19. Berek J, Novak E. Berek and Novak’s Gynecology, 14th ed. Philadelphia: Services; 2010. Lippincott Williams & Wilkins; 2007:548. 2. World Health Organization. UNAIDS Report on the Global AIDS 20. Olmsted SS, Padgett JL, Yudin AI, Whaley KJ, Moench TR, Cone RA. Epidemic. Geneva, Switzerland: WHO Press; 2010. Diffusion of macromolecules and virus-like particles in human cervical 3. Langbein L, Grund C, Kuhn C, Praetzel S, Kartenbeck J, Brandner JM, mucus. Biophys J 2001; 81:1930–1937. Moll I, Franke WW. Tight junctions and compositionally related 21. Harada N, Iijima S, Kobayashi K, Yoshida T, Brown WR, Hibi T, Oshima junctional structures in mammalian stratified epithelia and cell cultures A, Morikawa M. Human IgGFc binding protein (FcgammaBP) in colonic derived therefrom. Eur J Cell Biol 2002; 81:419–435. epithelial cells exhibits mucin-like structure. J Biol Chem 1997; 4. Marchiando AM, Graham WV, Turner JR. Epithelial barriers in 272:15232–15241. homeostasis and disease. Annu Rev Pathol 2010; 5:119–144. 22. Ghosh M, Fahey JV, Shen Z, Lahey T, Cu-Uvin S, Wu Z, Mayer K, 5. Meng W, Takeichi M. : molecular architecture and Wright PF, Kappes JC, Ochsenbauer C, Wira CR. Anti-HIV activity in regulation. Cold Spring Harb Perspect Biol 2009; 1:1–13. cervical-vaginal secretions from HIV-positive and -negative women 6. Green KJ, Simpson CL. Desmosomes: new perspectives on a classic. J

correlate with innate antimicrobial levels and IgG antibodies. PLoS One Downloaded from https://academic.oup.com/biolreprod/article/85/1/97/2530426 by guest on 27 September 2021 Invest Dermatol 2007; 127:2499–2515. 2010; 5:e11366. 7. Gorodeski GI. Estrogen decrease in tight junctional resistance involves 23. Anderson DJ. Genitourinary immune defense. In: Holmes KK, Sparling matrix-metalloproteinase-7-mediated remodeling of occludin. Endocrinol- PF, Stamm WE, Piot P, Wasserheit JN, Corey L, Cohen MS, Watts DH ogy 2007; 148:218–231. (eds.), Sexually Transmitted Diseases, 4th ed. New York: McGraw-Hill; 8. Nusrat A, Turner JR, Madara JL. Molecular physiology and pathophys- 2008:271–288. iology of tight junctions. IV. Regulation of tight junctions by extracellular 24. Pudney J, Quayle AJ, Anderson DJ. Immunological microenvironments in stimuli: nutrients, cytokines, and immune cells. Am J Physiol Gastrointest the human vagina and cervix: mediators of cellular immunity are Liver Physiol Physiol 2000; 279:G851–G857. concentrated in the cervical transformation zone. Biol Reprod 2005; 9. Sawada N, Murata M, Kikuchi K, Osanai M, Tobioka H, Kojima T, Chiba 73:1253–1263. H. Tight junctions and human diseases. Med Electron Microsc 2003; 25. Johnson LG. Applications of imaging techniques to studies of epithelial 36:147–156. tight junctions. Adv Drug Deliv Rev 2005; 57:111–121. Downloaded from www.biolreprod.org. 10. Shen L, Weber CR, Turner JR. The tight junction protein complex undergoes rapid and continuous molecular remodeling at steady state. J 26. de Boer CJ, van Dorst E, van Krieken H, Jansen-van Rhijn CM, Warnaar Cell Biol 2008; 181:683–695. SO, Fleuren GJ, Litvinov SV. Changing roles of and catenins 11. Yeo NK, Jang YJ. Rhinovirus infection-induced alteration of tight junction during progression of squamous intraepithelial lesions in the uterine and adherens junction components in human nasal epithelial cells. cervix. Am J Pathol 1999; 155:505–515. Laryngoscope 2010; 120:346–352. 27. Jeffers MD, Paxton J, Bolger B, Richmond JA, Kennedy JH, McNicol 12. Colegio OR, Van Itallie CM, McCrea HJ, Rahner C, Anderson JM. AM. E-cadherin and integrin expression in Claudins create charge-selective channels in the paracellular pathway invasive and in situ carcinoma of the cervix. Gynecol Oncol 1997; between epithelial cells. Am J Physiol Cell Physiol 2002; 283:C142– 64:481–486. C147. 28. Economopoulou M, Hammer J, Wang F, Fariss R, Maminishkis A, Miller 13. Lee JW, Lee SJ, Seo J, Song SY, Ahn G, Park CS, Lee JH, Kim BG, Bae SS. Expression, localization, and function of junctional adhesion DS. Increased expressions of claudin-1 and claudin-7 during the molecule-C (JAM-C) in human retinal pigment epithelium. Invest progression of cervical neoplasia. Gynecol Oncol 2005; 97:53–59. Ophthalmol Vis Sci 2009; 50:1454–1463. 14. Sobel G, Szabo I, Paska C, Kiss A, Kovalszky I, Kadar A, Paulin F, Schaff 29. Ostermann G, Weber KS, Zernecke A, Schroder A, Weber C. JAM-1 is a Z. Changes of cell adhesion and extracellular matrix (ECM) components ligand of the beta(2) integrin LFA-1 involved in transendothelial migration in cervical intraepithelial neoplasia. Pathol Oncol Res 2005; 11:26–31. of leukocytes. Nat Immunol 2002; 3:151–158. 15. Bouschbacher M, Bomsel M, Verronese E, Gofflo S, Ganor Y, Dezutter- 30. Higgins JM, Mandlebrot DA, Shaw SK, Russell GJ, Murphy EA, Chen Dambuyant C, Valladeau J. Early events in HIV transmission through a YT, Nelson WJ, Parker CM, Brenner MB. Direct and regulated interaction human reconstructed vaginal mucosa. AIDS 2008; 22:1257–1266. of integrin alphaEbeta7 with E-cadherin. J Cell Biol 1998; 140:197–210. 16. Nazli A, Chan O, Dobson-Belaire WN, Ouellet M, Tremblay MJ, Gray- 31. Guttman JA, Kazemi P, Lin AE, Vogl AW, Finlay BB. Desmosomes are Owen SD, Arsenault AL, Kaushic C. Exposure to HIV-1 directly impairs unaltered during infections by attaching and effacing pathogens. Anat Rec mucosal epithelial barrier integrity allowing microbial translocation. PLoS (Hoboken) 2007; 290:199–205. Pathog 2010; 6:e1000852. 32. Van Lint P, Libert C. Chemokine and cytokine processing by matrix 17. Vetrano S, Rescigno M, Cera MR, Correale C, Rumio C, Doni A, Fantini metalloproteinases and its effect on leukocyte migration and inflammation. M, Sturm A, Borroni E, Repici A, Locati M, Malesci A, et al. Unique role J Leukoc Biol 2007; 82:1375–1381. of junctional adhesion molecule-A in maintaining mucosal homeostasis in 33. Anderson DJ, Politch JA, Nadolski AM, Blaskewicz CD, Pudney J, Mayer inflammatory bowel disease. Gastroenterology 2008; 135:173–184. KH. Targeting Trojan Horse leukocytes for HIV prevention. AIDS 2010; 18. Zen K, Babbin BA, Liu Y, Whelan JB, Nusrat A, Parkos CA. JAM-C is 24:163–187.