Distinct Proinflammatory Host Responses to Neisseria gonorrhoeae Infection in Immortalized Human Cervical and Vaginal Epithelial Cells

Abstract

In this study we utilized immortalized morphologically and functionally distinct epithelial cell lines from normal human endocervix, ectocervix, and vagina to characterize gonococcal epithelial interactions pertinent to the lower female genital tract. Piliated, but not nonpiliated, N. gonorrhoeae strain F62 variants actively invaded these epithelial cell lines, as demonstrated by an antibiotic protection assay and confocal microscopy. Invasion of these cells by green fluorescent protein-expressing gonococci was characterized by colocalization of gonococci with F actin, which were initially detected 30 min postinfection. In all three cell lines, upregulation of interleukin 8 (IL-8) and IL-6, intercellular adhesion molecule 1 (CD54), and the nonspecific cross-reacting antigen (CD66c) were detected 4 h after infection with piliated and nonpiliated gonococci. Furthermore, stimulation of all three cell lines with gonococcal whole-cell lysates resulted in a similar upregulation of IL-6 and IL-8, confirming that bacterial uptake is not essential for this response. Increased levels of IL-1 were first detected 8 h after infection with gonococci, suggesting that the earlier IL-8 and IL-6 responses were not mediated through the IL-1 signaling pathway. The IL-1 response was limited to cultures infected with piliated gonococci and was more vigorous in the endocervical epithelial cells. The ability of gonococci to stimulate distinct proinflammatory host responses in these morphologically and functionally different compartments of the lower female genital tract may contribute directly to the inflammatory signs and symptoms characteristic of disease caused by N. gonorrhoeae.Cervicovaginal epithelial cells are increasingly recognized as active moderators of both innate and acquired immune functions in the genital tract via the production of a variety of proinflammatory and anti-inflammatory mediators. Increased levels of the cytokines interleukin 1 (IL-1), tumor necrosis factor alpha (TNF-␣), and IL-6 and the chemokines RANTES, MIP-1␣, and MIP-1␤ in cervicovaginal secretions have been associated with human immunodeficiency virus type 1 infection and bacterial vaginosis; however, little is known about the role of locally produced proinflammatory mediators in the pathogenesis of bacterial vaginitis and cervicitis (10). We recently developed three epithelial cell lines from normal human vagina, ectocervix, and endocervix, immortalized by expression of the E6 and E7 genes of human papillomavirus type 16 (8). The morphological and immunocytochemical characteristics of the immortalized lines closely resembled those of their tissues of origin and primary cultures, and all three differed significantly from the HeLa cervical adenocarcinoma cell line, the most commonly used cell line derived from the human female lower genital tract mucosa. The three cell lines constitutively produced distinctive arrays of cytokines (IL-1, IL-6, IL-7, macrophage colony-stimulating factor, and transforming growth factor ␤) and chemokines (IL-8 and RANTES),...

TREM-2 binds to lipooligosaccharides of Neisseria gonorrhoeae and is expressed on reproductive tract epithelial cells

“…Cervical carcinoma cell lines ME-180 and HeLa, monocytic cell line THP-1, and three HPV16/ E6E7 immortalized reproductive tract epithelial cell lines, endocervical End1/E6E7, ectocervical Ect1/E6E7, and vaginal Vk2/E6E7, were obtained from the American Type Culture Collection (Manassas, VA) and have been characterized previously (7,10,12). The E6E7 cell lines were maintained in keratinocyte serum-free medium (Invitrogen, Carlsbad, CA) supplemented with 0.05mg/ml bovine pancreatic extract, 0.1 ng/ml recombinant human epidermal growth factor, and 0.44 μg/ml CaCl 2 .…”

“…In particular, gonococci engage innate immune receptors TLR4 and TLR2 with lipooligosaccharide (LOS) engaging TLR4, and PorB and lipoprotein Lip functioning as agonists for TLR2 (7)(8)(9). In urogenital epithelial cell lines, gonococcal infection induces upregulation of a variety of proinflammatory and inflammatory cytokines, including TNF-α, IL-1β, IL-6, and IL-8 (10)(11)(12). In experimental infection, the levels of the same four cytokines have been found to be elevated in both the urine and plasma of men after gonococcal challenge (13).…”

Response toNeisseria gonorrhoeaeby Cervicovaginal Epithelial Cells Occurs in the Absence of Toll-Like Receptor 4-Mediated Signaling

“…We have used this model to study the downstream effects of cell signaling following Neisseria gonorrhoeae infection. Our previous investigations showed that all three epithelial cell lines respond to N. gonorrhoeae by up-regulation of cytokines and adhesion molecules and that these responses are independent of bacterial internalization and IL-1 production (10). However, the key components of the signaling pathways involved in the inflammatory responses of cervicovaginal epithelial cells to Gram-negative bacteria have yet to be identified.…”

Variability in human semen content and its potential effects in the female reproductive tract

“…Because this region is exposed to a lower level of stimuli relative the vaginal epithelium, the tolerance of the epithelium might be lower and its responsiveness to doi: 10.7243/2054-0841-4-1 insults correspondingly higher [27]. Studies have shown that even in the cervix there are regional differences, with the endocervix (higher up in the FRT) having a greater response to foreign stimuli [29]. Perhaps this robust response is due to the fact that the upper cervical region sees fewer foreign stimuli than the lower region and stands poised to provide a more effective response to stop colonization of the upper cervix [29].…”

“…Studies have shown that even in the cervix there are regional differences, with the endocervix (higher up in the FRT) having a greater response to foreign stimuli [29]. Perhaps this robust response is due to the fact that the upper cervical region sees fewer foreign stimuli than the lower region and stands poised to provide a more effective response to stop colonization of the upper cervix [29].…”