Mucosal surfaces are the first line of defense against potentially pathogenic microorganisms. Mucosal FRT epithelial cells have the capacity to recognize and respond immediately to pathogens and can rapidly block infection from being established. In a prior study 19
, we demonstrated that human uterine epithelial cells in culture produce an anti-bacterial factor(s) that is equally effective in inhibiting the growth of a gram-positive (S. aureus
) and a gram-negative bacterium (E. coli
). To explore the possibility that secretions from epithelial cells along the entire FRT have antimicrobial activity, cultured epithelial cells from the Fallopian tubes, uterus, endocervix and ectocervix were grown to confluence in cell inserts, and assessed for their ability to secrete a substance(s) into the apical compartment that would inhibit bacterial and fungal growth, as well as HIV-1 infectivity. We found that epithelial cell conditioned media from all FRT sites examined inhibited growth of C. albicans
(yeast and hyphae) and N. gonorrhoeae
. In addition, FRT epithelial secretions were effective in significantly reducing HIV infection of TZM-bl cells. Just as important, FRT epithelial cell secretions had no effect on the growth of the urogenital commensal L. crispatus
. Although vaginal squamous cells were not available owing to the hysterectomy specimens provided, squamous cells from ectocervical tissues were included in this study. As seen in , and , secretions from these cells had inhibitory activities comparable to that seen with columnar epithelial cells from the upper FRT.
It is well recognized that C. albicans
is both a commensal as well as a pathogen in the lower female reproductive tract. As a part of its life cycle C. albicans
exists in a nonpathogenic yeast form as well as a potential pathogen during the hyphal stage. Recognized as a common component of the digestive and genital floras, C. albicans
has the potential to cause superficial as well as disseminated infections in response to host immune system changes, oral contraceptive use or microflora alterations 4,41
. In contrast to the vagina, the upper FRT lacks C. albicans
receptors and an appropriate nutritional status that most likely confer vaginal tissue tropism. Our studIes demonstrate that FRT epithelial cells secrete a spectrum of antimicrobials that have anti-C. albicans
(yeast and hyphal forms) activity. An unexpected finding in our study was the differential effect of epithelial cell secretions on the commensal form of C. albicans
(yeast) and Lactobacillus. Whereas all secretions tested inhibited the commensal form of C. albicans
, none had an inhibitory effect on Lactobacillus. What is clear is that factors in epithelial secretions selectively discriminate between these two commensals.
Analysis of conditioned media led to the identification of a spectrum of antimicrobials, chemokines and cytokines secreted by uterine epithelial cells. We have analyzed the conditioned media for biological activity and identified MCP-1, IL-8, GM-CSF and SLPI based on either chemotaxis or antimicrobial assays 13,19,42
. Many of these innate immune factors are multi-functional in that they exhibit both chemokine and antimicrobial activity. For example, MIP3α is known to be chemotactic for immature dendritic cells, T cells and B cells 43,44
. This chemokine was found to inhibit both gram negative and gram positive bacteria 45
. Our recent studies extend these findings by demonstrating that MIP3α as well as Trappin-2/Elafin are potent antiviral molecules capable of blocking HIV-1 infection of target cells 34,35
. The extent to which other molecules secreted by epithelial cells throughout the FRT have cytokine and chemokine activity, as well as potent anti-bacterial, anti-fungal and anti-viral activity, remains to be determined.
In other studies in which we identified rElafin and rCCL20/MIP3α as novel antimicrobials against HIV 34,35
, we found that neutralization of recombinants was possible with complete reversal of anti-HIV-1 activity. However, using these same antibodies, we did not reverse the anti-HIV-1 activity in uterine apical secretions containing Elafin and MIP3α, present at concentrations that should be completely blocked. We and others have previously shown FRT secretions contain a family of antimicrobials, which have anti-HIV-1 activity 34,35
. Since Elafin and MIP3α are two of the many molecules present in these secretions, neutralization would not be measurable as a separate entity.
To the best of our knowledge, this is the first study to demonstrate that epithelial cells from throughout the human FRT, under constitutive conditions, inhibit a wide range of organisms that both compromise women's reproductive health and are potentially life threatening. Others have shown that commensal microorganisms are constitutively present in the lower FRT as part of a genital tract ecosystem that prevents the establishment of infections by pathogens via a variety of mechanisms (adherence, pH, bacteriocins, etc.). Our studies suggest that a dynamic balance exists in the upper FRT between innate immune epithelial cell factors and commensals in maintaining a healthy homeostatic microenvironment in the lower FRT. Unlike antibiotics, which can kill commensal bacteria in the lower FRT and thereby lead to opportunistic infections, endogenous epithelial factors are constitutively produced to provide long-term protection against pathogens. The mechanism(s) by which Lactobacillus is resistant to endogenous antimicrobials remains elusive, and is further complicated by the recent discovery that Lactobacillus stimulates β-defensin secretion by enterocytes 46
. Commensals and epithelial cells act separately as well as in conjunction to protect the FRT from infection. This symbiotic relationship is particularly appropriate when one considers that secretions from the upper tract flow to the lower tract, while lower tract secretions, as well as sperm, move into the upper tract 47
What remains to be determined is how genital tract secretions exert their inhibitory effects on bacterial, fungal and viral pathogens. Antimicrobials in the defensin family have been shown to cause pore formation in bacterial cell walls resulting in death of the bacterium. RANTES blocks HIV-1 infection by binding to the HIV co-receptor CCR5, preventing viral attachment, while Trappin-2/Elafin and MIP3α inhibit HIV-1 infection by interacting with the virus 34,35
. SLPI, which inhibits Candida 29,48
, HIV 26,30,49,50
and bacteria 51
, has multiple mechanisms to protect the FRT. For example, SLPI inhibits HIV-1 infection by binding to annexin-1 on the cell surface, and by inhibiting NFkB stimulation for immune activation that promotes HIV infection. CCL28 inhibits bacteria and Candida 52
and possibly HIV infection by binding to CCR3, which has been shown to be an alternative coreceptor for HIV-1 53
. Regardless of mechanism, it is clear that epithelial cells secrete a spectrum of antimicrobials that are protective against microbial pathogens.
Recognizing that sexual transmission is almost exclusively initiated by HIV-1 variants with tropism for the CCR5 co-receptor (R5 viruses) 54
, we tested epithelial secretions and found that Fallopian tube, uterine, cervix and ectocervix epithelial secretions inhibited BaL (R5) virus. To the best of our knowledge, this is the first demonstration that secretions from epithelial cells throughout the human female reproductive tract have anti-HIV activity. Further studies are needed to identify the antimicrobial(s) responsible for inhibition of HIV and whether the antimicrobials secreted are cell and site specific, or produced by epithelial cells independent of their anatomical location (upper vs. lower FRT). In other studies, we have recently found that cervical vaginal secretions from both HIV (-) women and HIV (+) women who are healthy and not on anti-retrovirals (ARV) have anti-HIV activity against R5 HIV-1 55
. These findings suggest that antimicrobials produced by epithelial cells from throughout the reproductive tract play an important protective role in vivo against the transmission of HIV from men to women.
In conclusion, these studies demonstrate that normal female reproductive tract epithelial cells secrete a spectrum of antimicrobials in culture that have broad-spectrum activity against bacterial, fungal and viral pathogens. These results emphasize the central role of uterine epithelial cells in protecting the female reproductive tract from sexually transmitted diseases and opportunistic infections.