Our finding that monocytes were involved in the differential apoptosis observed for D/UW-3/Cx and CTD153 (Fig

Our finding that monocytes were involved in the differential apoptosis observed for D/UW-3/Cx and CTD153 (Fig. most common bacterial sexually transmitted infection in the world, with WHO estimates indicating that 90 million individuals are infected worldwide (48). Acute infection is often asymptomatic, but untreated or repeat infections can result in immunopathology including pelvic inflammatory disease (PID), chronic pelvic pain, ectopic pregnancy, and infertility. This pathology results from an aggressive host inflammatory response, and human studies have provided insight into potential inflammatory mediators of reproductive tract sequelae. A study examining endometrial biopsy specimens from women exhibiting signs of PID correlated infection with an inflammatory milieu consisting of both acute and chronic leukocyte populations, including neutrophils, plasma cells, and periglandular lymphoid follicles containing transformed lymphocytes (21). In patients with documented endometrial and oviduct infection with infection (47). Although studies in humans have not revealed a unilateral mediator of chlamydia-induced immunopathology, these reports indicate a potential role for neutrophils in causing tissue damage. Studies with the mouse model have supported this correlation by revealing that enhanced and/or prolonged neutrophil influx into the oviducts is associated with the development of hydrosalpinx (8, 42). In addition, mice deficient in the chemokine receptor CXCR2 display reduced acute inflammation and lower rates of hydrosalpinx, and strains of mice with elevated CXCL2 production exhibit worse disease (9, 24). Neutrophils likely contribute to pathology by releasing mediators that directly damage reproductive tract tissues, and neutrophil release of the proteolytic enzyme matrix metalloproteinase 9 (MMP9) has been implicated in the development of scarring and fibrosis of the murine oviduct after chlamydial infection (16, 38). In addition, mice deficient in NADPH oxidase (Nigg compared to results with CM3.1 and established potential mechanisms whereby this enhanced neutrophil response leads to increased pathology. MATERIALS AND METHODS Strains, cell lines, and culture conditions. The strains Nigg (provided by Roger Rank) and CM3.1, as β-Apo-13-carotenone D3 well as the strains D/UW-3/Cx and CTD153, used in this study, were previously described (31, 32). All chlamydial strains were propagated in L929 cells (30). Bacteria were titrated by plaque assay (32) or as inclusion-forming units (IFU) using fluorescently tagged anti-chlamydial lipopolysaccharide monoclonal antibody (Bio-Rad, Hercules, CA). Bacterial titers were confirmed using real-time PCR for chlamydial 16S rRNA (30). Live chlamydiae were used for all experiments. experiments comparing responses with D/UW-3/Cx and CTD153 were repeated with bacteria from at least two different preparations in order to account for variability in chlamydial stimulation resulting from isolation or titration techniques. Animals. Six- to eight-week-old female C3H/HeOuJ and C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The mice were between 8 and 12 weeks of age at the time of infection. Mice were given food and water in an environmentally controlled room with a cycle of 12 h of light and 12 h of darkness. All animal experiments were preapproved by the University of Pittsburgh Institutional Animal Care and Use Committee. Murine infection and monitoring. Seven days prior to infection, mice Il1a were subcutaneously injected with 2.5 mg of progesterone (Depo-Provera; Upjohn, Kalamazoo, MI) to synchronize mice in a state of anestrous and to facilitate β-Apo-13-carotenone D3 successful intravaginal infection (44). Mice were infected with 1 105 IFU of Nigg or CM3.1 intravaginally. Mice were β-Apo-13-carotenone D3 monitored for cervicovaginal shedding (20), and IFUs were calculated as described previously (8). The bacterial burden was measured in the oviducts by serially diluting a 100-l aliquot of the homogenized oviducts in 1 phosphate-buffered saline (PBS) for titration using a plaque assay (32). Processing of oviducts for flow cytometry. Oviducts were processed for flow cytometric analysis as previously described (41). Briefly, oviducts were harvested into 1 ml of medium (RPMI plus 1% fetal bovine serum [FBS]) and minced with scissors. For measurement of cytokines and bacterial burden, 100 l was removed and stored at ?80C until analysis. The tissue was repeatedly.