Phenotype and susceptibility to HIV infection of CD4+ Th17 cells in the human female reproductive tract.

Prevention of sexual acquisition of HIV in women requires a substantial increase in our knowledge about HIV-target cell availability and regulation in the female reproductive tract (FRT). In this study, we analyzed the phenotype and susceptibility to HIV infection of CD4(+) T cell in the endometrium (EM), endocervix (END), and ectocervix (ECT) of the FRT. We found that T helper type 17 (Th17) cells represent a major subset in FRT tissues analyzed and that Th17 cells were the main CD4(+) T-cell population expressing C-C motif chemokine receptor 5 (CCR5) and CD90. In premenopausal women, CD4(+) T cells and Th17 cells, in particular, were significantly lower in EM relative to END and ECT. Th17 cells were elevated in EM from postmenopausal women relative to premenopausal tissues but not changed in END and ECT. Susceptibility of CD4(+) T cells to HIV infection measured as intracellular p24 was lowest in the EM and highest in the ECT. Additionally, we found that Th17 cells co-expressing CCR5 and CD90 were the most susceptible to HIV infection. Our results provide valuable information for designing preventive strategies directed at targeting highly susceptible target cells in the FRT.

Innate and adaptive immunity at mucosal surfaces of the female reproductive tract: stratification and integration of immune protection against the transmission of sexually transmitted infections.

This review examines the multiple levels of pre-existing immunity in the upper and lower female reproductive tract. In addition, we highlight the need for further research of innate and adaptive immune protection of mucosal surfaces in the female reproductive tract. Innate mechanisms include the mucus lining, a tight epithelial barrier and the secretion of antimicrobial peptides and cytokines by epithelial and innate immune cells. Stimulation of the innate immune system also serves to bridge the adaptive arm resulting in the generation of pathogen-specific humoral and cell-mediated immunity. Less understood are the multiple components that act in a coordinated way to provide a network of ongoing protection. Innate and adaptive immunity in the human female reproductive tract are influenced by the stage of menstrual cycle and are directly regulated by the sex steroid hormones, progesterone and estradiol. Furthermore, the effect of hormones on immunity is mediated both directly on immune and epithelial cells and indirectly by stimulating growth factor secretion from stromal cells. The goal of this review is to focus on the diverse aspects of the innate and adaptive immune systems that contribute to a unique network of protection throughout the female reproductive tract.

Epithelial cell secretions from the human female reproductive tract inhibit sexually transmitted pathogens and Candida albicans but not Lactobacillus.

Female reproductive tract (FRT) epithelial cells protect against potential pathogens and sexually transmitted infections. The purpose of this study was to determine if epithelial cells from the upper FRT secrete antimicrobials that inhibit reproductive tract pathogens that threaten women's health. Apical secretions from primary cultures of Fallopian tube, uterine, cervical, and ectocervical epithelial cells were incubated with Neisseria gonorrhoeae, Candida albicans (yeast and hyphal forms), human immunodeficiency virus 1 (HIV-1), and Lactobacillus crispatus before being tested for their ability to grow and/or infect target cells. Epithelial cell secretions from the upper FRT inhibit N. gonorrhoeae and both forms of Candida, as well as reduce HIV-1 (R5) infection of target cells. In contrast, none had an inhibitory effect on L. crispatus. An analysis of cytokines and chemokines in uterine secretions revealed several molecules that could account for pathogen inhibition. These findings provide definitive evidence for the critical role of epithelial cells in protecting the FRT from infections, without comprising the beneficial presence of L. crispatus, which is part of the normal vaginal microflora of humans.

Estradiol selectively regulates innate immune function by polarized human uterine epithelial cells in culture.

The goal of this study was to examine the role of E(2) in regulating innate immune protection by human uterine epithelial cells (UECs). Recognizing that UECs produce cytokines and chemokines to recruit and activate immune cells as well as viral and bacterial antimicrobials, we sought to examine the effect of E(2) on constitutive and Toll-like receptor (TLR) agonist (lipopolysaccharide (LPS) and poly (I:C))-induced immune responses. The secretion by polarized UECs in culture of interleukin (IL)-6, macrophage inhibitory factor (MIF), and secretory leukocyte protease inhibitor (SLPI) was examined as well as the mRNA expression of human beta-defensin-2 (HBD2), tumor necrosis factor (TNF)-alpha, IL-8, and nuclear factor (NF)-kB. When incubated with E(2) for 24-48 h, we found that E(2) stimulated UEC secretion of SLPI (fourfold) and mRNA expression of HBD2 (fivefold). Moreover, when antibacterial activity in UEC secretions was measured using Staphylococcus aureus, E(2) increased the secretion of soluble factor(s) with antibacterial activity. In contrast, E(2) had no effect on constitutive secretion of proinflammatory cytokines and chemokines by UECs but completely inhibited LPS- and poly (I:C)-induced secretion of MIF, IL-6, and IL-8. Estradiol also reversed the stimulatory effects of IL-1beta on mRNA expression of TNF-alpha, IL-8, and NF-kB by 85, 95, and 70%, respectively. As SLPI is known to inhibit NF-kB expression, these findings suggest that E(2) inhibition of proinflammatory cytokines may be mediated through SLPI regulation of NF-kB. Overall, these findings indicate that the production of cytokines, chemokines, and antimicrobials by UECs are differentially regulated by E(2). Further, it suggests that with E(2) regulation, epithelial cells that line the uterine cavity have evolved immunologically to be sensitive to viral and bacterial infections as well as the constraints of procreation.

Secretion of cytokines and chemokines by polarized human epithelial cells from the female reproductive tract.

BACKGROUND: Pro-inflammatory chemokines that attract and cytokines that activate immune cells contribute to normal physiological homeostasis in the female reproductive tract, and are needed to deal effectively with potential pathogenic microbes. Mucosal epithelial cells are capable of producing these factors that communicate with cells of the innate and adaptive immune systems. METHODS: Epithelial cells from Fallopian tube, endometrium and endocervix were isolated and grown to high transepithelial resistance in cell inserts from seven patients who had hysterectomies. Interleukin (IL)-8, IL-6, granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-alpha (TNF-alpha) and macrophage inflammatory peptide-1beta (MIP-1beta) were assessed by Luminex bead analysis or enzyme-linked immunosorbent assay (ELISA) in epithelial cell conditioned media from the apical and basolateral compartments. RESULTS: With the exception of MCP-1, the seven chemokines/cytokines constitutively produced by the polarized epithelial cells were preferentially secreted apically. A concentration pattern was found in all cases, with IL-8 and IL-6 produced in the greatest quantity. CONCLUSIONS: The concentrations of IL-8, IL-6, G-CSF and MCP-1 are similar to the levels found in reproductive tract fluids of patients with infection. The constitutive secretion and compartmentalization of large quantities of bioactive chemokines and cytokines provide additional evidence for the role of epithelial cells as gatekeepers of innate immune protection in the female reproductive tract.

Antigen-presenting cells in the human female reproductive tract: analysis of antigen presentation in pre- and post-menopausal women.

PROBLEM: To determine whether cells in the female reproductive tract (FRT) are functionally capable of presenting antigen to T cells. METHOD OF STUDY: Analysis was done by determining the proliferation of purified autologous T cells to antigen, following co-incubation with non-proliferating cell suspensions isolated from the uterus and prepared by enzymatic digestion of reproductive tract tissues from hysterectomy patients with benign disease. RESULTS: All uterine preparations analyzed were functionally capable of presenting antigen; the ability to present antigen was independent of pre- and post-menopausal status. In contrast, some, but not all, tissues from the ovary, Fallopian tube, cervix, and vagina were capable of presenting antigen. CONCLUSION: These results suggest that the human FRT is an inductive site for immune responses. Regulation of antigen presentation in the reproductive tract may be important for protection against sexually transmitted diseases.

Regulation by human uterine cells of PBMC proliferation: influence of the phase of the menstrual cycle and menopause.

To determine the influence of human uterine cells recovered at different stages of the menstrual cycle and following menopause on the proliferation of peripheral blood mononuclear cells (PBMC), whole cell suspensions of uterine tissues were co-cultured with autologous and heterologous PBMC. PBMC proliferation in response to tetanus toxoid (TT) or Con A was inhibited by uterine endometrial cells and was dependent on the phase of the menstrual cycle. Inhibition by cells from the proliferative phase was significantly greater than by cells from the secretory phase. Uterine cells isolated from post-menopausal women also inhibited proliferation of PBMC. Cell fractionation studies indicated that epithelial cells are the primary source of uterine inhibitory activity. When epithelial cells and PBMC were cultured in separate compartments, epithelial cells released a soluble factor(s) that inhibited the PBMC proliferation. These results suggest that uterine epithelial cells produce cytokines that down-regulate the proliferation of PBMC in response to antigens and mitogens. This may be important for the control of uterine immune responses, as well as the growth of the reproductive tract in preparation for implantation during the secretory phase of the menstrual cycle.

Secretory component production by polarized epithelial cells from the human female reproductive tract.

At mucosal surfaces, the polymeric Ig receptor (pIgR) is responsible for transporting polymeric IgA across epithelial cells. The purpose of this study was to determine whether normal epithelial cells from the female reproductive tract form tight junctions and produce secretory component, the external domain of the pIgR. Uterine, cervical and vaginal tissues from women at different stages of the menstrual cycle and following menopause were used to prepare purified epithelial cell sheets, which were cultured in cell chambers. Transepithelial resistance was measured and the media from apical and basolateral compartments assayed for secretory component. Secretory component produced by uterine epithelial cells accumulated preferentially in apical compartment and correlated with increased transepithelial resistance. Seeding as epithelial sheets at 1 x 10(6) cells/cm2 of matrix coated cell chambers was required for growth. Epithelial cells from endo-cervix and ecto-cervix, but not the vagina, also showed preferential production and release of secretory component into the apical chamber. In conclusion, normal epithelial cells from the human female reproductive tract grow to confluence, become polarized and produce secretory component. Our results suggest that uterine and cervical epithelial cells play a key regulatory role in the control of IgA transcytosis from tissue into secretions.

Collagenase production by synovial fibroblasts treated with phorbol myristate acetate.

A series of intracellular events occurring after treatment of rabbit synovial fibroblasts with 0.01 micrograms/ml phorbol myristate acetate (PMA) were measured. Ten minutes after addition of PMA, there was a temporary increase in intracellular cyclic AMP levels, followed by a transient decrease in incorporation of 3H-thymidine into DNA. Approximately 500 ng/mg cell protein of PGE2 were found in culture medium from the 12- to 24-hour incubation period, but significant collagenase was not detectable until 24 to 36 hours. Treatment with aspirin or indomethacin abolished PGE2 production but did not affect collagenase levels. Production of enzyme was associated with a cessation of cell proliferation, measured by protein content/culture and cell number. No enzyme was detectable in untreated cultures. Synovial fibroblasts treated with phorbol myristate acetate may provide a good model for studies on the mechanism of induction of collagenase production.

Regulation of bradykinin-induced cyclic amp response by quinacrine and prostaglandin E2 and F2 alpha in human synovial fibroblasts.

Bradykinin induces an increment in intracellular cyclic AMP concentrations of human synovial fibroblasts and evokes the release of [3H]arachidonic acid and [3H]-E prostaglandins from human synovial fibroblasts pre-labeled in their phospholipids. Both these bradykinin-induced reactions are inhibited by quinacrine, an inhibitor of phospholipase A activity. The cyclic AMP response of human synovial fibroblasts to bradykinin is potentiated by prostaglandin E2 and inhibited by prostaglandin F2 alpha. These data emphasize the critical role of the prostaglandin system in reactions induced by bradykinin and suggest mechansims by which inflammatory reactions due to bradykinin may be modulated.

Differential antibody forming cell response in mice due to levamisole treatment.

The administration of varying doses of levamisole on the antibody forming cell (AFC) response of C57BL6/J mice to sheep red blood cells was investigated. Doses of 0.5 and 2.5 mg/kg potentiated the AFC response, while a dose of 5 mg/kg levamisole blocked the AFC response. Injection of the latter does of levamisole one day after antigen exposure did not inhibit the AFC response. Levamisole did not alter the basal response to sheep red blood cells of otherwise untreated mice.

The effect of 7-oxa-13 prostynoic acid on the mechanism of action of bradykinin in human synovial fibroblasts.

Bradykinin, a potent inflammatory mediator, induces an increment in intracellular cyclic AMP concentrations of human synovial fibroblasts and evokes the synthesis and release of 3H-arachidonic acid and 3H-E prostaglandins from these cells pre-labeled in their phospholipids. Fetal calf serum in the media also stimulates the synthesis and release of these labeled lipids from pre-labeled human synovial fibroblasts and potentiates the bradykinin-induced cyclic AMP response. The PGE1 analogue, 7-oxa-13 prostynoic acid, completely abrogates both the bradykinin-induced cyclic AMP response and the bradykinin- and fetal calf serum-evoked release of labeled E-prostaglandins from pre-labeled cells. In serum-free media, the prostaglandin antagonist stimulated the release of 3H-arachidonic acid from pre-labeled human synovial fibroblasts and did not inhibit the bradykinin-induced release of this lipid.

Human synovial fibroblasts: the relationships between cyclic AMP, bradykinin, and prostaglandins.

Human synovial fibroblasts in culture respond to bradykinin (8 X 10(-9) M) with an increment in intracellular cyclic AMP concentration. These bradykinin (BK) concentrations are comparable to levels of the nonapeptide found in pathological synovial effusions. The cyclic AMP response to BK is enhanced by a heat stable factor(s) in fetal calf serum (FCS) and by the addition of arachidonic acid (AA) to monolayer cultures incubated in serum-free media. Synovial fibroblasts initially treated with BK are refractory to rechallenge with this agent as measured by the absence of an increment in cyclic AMP. These BK refractory cells do respond with significant increment in cyclic AMP to challenge with prostaglandin E1 (PGE1). Cells that have become refractory to PGE1 stimulation respond to BK. this suggests that a receptor or activator system different from the one for PGE1 and PGE2 exists for BK. When both BK and PGE1 are incubated together with synovial fibroblasts, the cyclic AMP response elicited is more than additive as compared to the response of each hormone separately. Indomethacin (IM) inhibits the BK evoked cyclic AMP response unless cell cultures are pretreated with PGE1. The PGE1 analog, 7-oxa-13-prostynoic acid, is a better inhibitor of the cyclic AMP response induced by BK than by PGE1. BK does not elicit a cyclic AMP response solely by elaborating PGE1, yet the prostaglandin pathway and its products seem to have a role in the degree of the cyclic AMP response to BK challenge.

Hydrocortisone inhibition of the bradykinin activation of human synovial fibroblasts.

Human synovial fibroblasts in culture respond to bradykinin with a 20-fold increment in intracellular cyclic AMP concentrations, however bradykinin does not directly activate adenylate cyclase activity in a particulate fraction derived from these cells. Bradykinin evokes a release of labeled arachidonic acid and prostaglandins E and F from synovial fibroblasts pre-labeled with 3H-arachidonic acid. Hydrocortisone inhibits the bradykinin induced increment in cyclic AMP and the release of arachidonic acid and prostaglandins E and F from synovial fibroblasts. Indomethacin, which also inhibits the cyclic AMP response to bradykinin, has no effect on the release of arachidonic acid from synovial fibroblasts. Indomethacin does, however, inhibit the quantity of prostaglandins released into the medium. These studies support the hypothesis that bradykinin does not activate human synovial fibroblast adenylate cyclase, but presumably activates a phospholipase whose products in turn result in the synthesis of prostaglandins. These and other investigations also suggest that a product(s) of the prostaglandin pathway causes the increment in cyclic AMP.

Prostaglandin synthetase activity and hormone responsiveness in normal and SV40 transformed WI-38 fibroblasts.

Prostaglandin (PG) synthetase activity and selective hormone responsiveness were examined in normal and SV40 transformed WI-38 fibroblasts (VA13-2RA). The transformed VA13-2RA cells have significantly reduced rates of PGE1, PGE2, PGF1alpha and PGF2alpha synthesis as compared to the normal WI-38 fibroblast. The transformed cell in contrast to the normal cell hyperresponds to stimulation by L-epinephrine (10 muM) and PGE1 (8.5 muM) but is unresponsive to bradykinin (BK) as measured by the accumulation of intracellular cyclic AMP. Indomethacin treatment does not significantly alter the PGE1 and L-epinephrine (EPI) responsiveness of the normal WI-38 fibroblast, however it abolishes the BK response in these cells. These results provide further evidence for the dependency of cell activation by bradykinin on the PG synthetase system. No experimental data was found to support the role of PGs as negative regulators of PGE1 and EPI responsiveness in the WI-38 fibroblast. Using the VA13-2RA cells, limited attempts to recover PG synthetase activity comparable to that found in normal WI-38 cells were unsuccessful. The VA13-2RA cell and its normal counterpart represent models for investigating the role of PGs in cell function and the mechanism of BK activation and its effect on cell metabolism.

Human synoviocytes: activation and desensitization by prostaglandins and 1-epinephrine.

The human synoviocyte increases its intracellular adenosine 3':5'-cyclic monophosphate (cAMP) concentration significantly after incubation with prostaglandin E1, prostaglandin E2, or l-epinephrine. The cells can be desensitized to these same hormones. Hormone-induced desensitization is receptor site specific and associated with a significant increase in intracellular 3':5'-cyclic AMP phosphodiesterase (EC 3.1.4. 17; 3':5'-cyclic AMP 5'-nucleotidohydrolase) activity, whereas cAMP-induced desensitization is not hormone specific.

PGE1-mediated cyclic AMP refractoriness: effects of cycloheximide and indomethacin.

Human synoviocytes in culture respond to prostaglandin E1 (PGE1) by increasing their intracellular concentration of cyclic AMP. Readdition of PGE1 to cells previously treated with PGE1 elicits no change in the intracellular concentration of cyclic AMP. This refractory state is partially prevented by the inhibitors of protein synthesis, puromycin (PM) and cycloheximide (CH). Indomethacin (IM), which reduces angiotensin tachyphylaxis, does not prevent the occurrence of refractoriness to PGE1 with respect to accumulation of cyclic AMP. This agent does alter the release of cyclic AMP from human synovial cells. We postulate that other factors, independent of new protein synthesis, are necessary for the development of the complete PGE1 refractory state in these cells.