Uterine epithelial cell release of MIP3alpha, TNFalpha and TGFbeta in response to Escherichia coli, Lactobacillus rhamnosus and pathogen associated molecular patterns (PAMP) and the influence of estradiol on cytokine release

The research presented in this thesis tested the hypothesis that uterine epithelial cells (UEC) are selectively responsive to bacteria and pathogen associated molecular patterns (PAMP) and that responses are influenced by estradiol (E2). We developed a model system to define microbially elicited epithelial cell basolateral cytokine release, in which UEC from adult rats were grown to confluence in cell culture inserts and monitored for polarization as determined by transepithelial resistance (TER). Polarized UEC were treated apically with live or heat-killed (HK) Escherichia coli ( E. coli) or Lactobacillus rhamnosus (L. rhamnosus) prior to collection of basolateral media after 24 hr incubation. Co-culture of polarized UEC with live E. coli had no effect on UEC TER. However, in response to live E. coli exposure, UEC release of MIP3alpha and TNFalpha increased at a time when release of TGFbeta decreased. Incubation of UEC with HK-E. coli stimulated release of MIP3alpha and TNFalpha, without affecting TER or TGFbeta. While UEC were responsive to E. coli, live or HK L. rhamnosus had no effect on TER or cytokine release. In response to LPS, LTA and Pam3Cys, MIP3alpha and TNFalpha, release increased. Time course studies of MIP3alpha and TNFalpha, production in response to PAMP indicated that MIP3alpha release peaked 2 to 4 hours after treatment whereas TNFalpha, release was gradual. To determine the effects of E2 on MIP3alpha and TNFalpha release, UEC were cultured in media with stripped fetal bovine serum (F12K-SS) which contains estrogen concentrations below the level of detection. When UEC cultures were pre-cultured in F12K-SS for 24 hr followed by 24 hr treatment in F12K-SS with estradiol, constitutive release of both MIP3alpha and TNFalpha, was inhibited. However when UEC were treated with PAMP, E2 stimulated MIP3alpha release at a time when TNFalpha release was inhibited relative of UEC treated with PAMP alone. The estrogen receptor antagonist ICI 182,780 failed to reverse constitutive-E2-inhibitory effects on MIP3alpha and TNFalpha. In contrast, treatment with ICI 182,780 reversed the stimulatory effect of E2 on PAMP-stimulated MIP3alpha release. Taken together, these findings suggest that UEC responses to bacteria are selective and that estradiol regulates the release of MIP3alpha and TNFalpha by UEC.