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An in vitro cell culture system was used to study the effect ofinterferon V(IFN-y) on Chiamydia trachomatis growth and differentiation. The effect of IFN-y on chlamydiae was dose-dependent. IFN-y at 2 ng/ml completely inhibited chlamydial growth and differentiation; however, persistent infection was established when chlamydiae were cultured with IFN-y at 0.2 ng/ml. Persistent infection was characterized by the development of noninfectious atypical chlamydial forms from which infectious progeny could be recovered only when IFN-y was removed from the culture system. defined, but previous studies demonstrate that treatment of host cells with interferon 'y (IFN-'y) before infection with chlamydiae results in the formation of atypical RBs that fail to differentiate into infectious progeny (8-12). Because persistent infections have been proposed as a source of antigen for the stimulation of immunopathology that leads to blindness and infertility, we evaluated whether infectious chlamydiae could be rescued from the aberrant RB forms that follow IFN-y treatment and whether the levels of important chlamydial immunogens were altered during abnormal growth.MATERIALS AND METHODS Organism. C. trachomatis serovar A/Har-13 was grown in HeLa 229 cells, and EBs were purified by discontinuous density centrifugation in Renografin (Squibb) (13).Preparation of IFN-y-Treated Cultures and Assay for Infectivity. HeLa 229 cells in minimal essential medium with 10% fetal bovine serum (MEM-10) were plated at a density of 1.5 x 105 cells per well in 24-well culture plates and maintained at 37°C in 5% CO2. Eighteen to 24 hr later the cell monolayers were washed once with Hanks' balanced salt solution (HBSS) and then treated for 15 min at room temperature with HBSS containing DEAE-dextran (45 ,g/ml), followed by two washes with HBSS. The final wash was removed and replaced with 0.2 ml of 0.25 M sucrose/10 mM sodium phosphate/5 mM L-glutamic acid (SPG), pH 7.2, containing 3 x 105 inclusion-forming units (IFU) of C. trachomatis serovar A (HAR-13), and incubated for 2 hr at 37°C on a rocker platform. Two hours after infection the inoculum was removed and replaced with 0.5 ml of either MEM-10 or MEM-10 containing recombinant human IFN-y at 0.05, 0.2, or 2.0 ng/ml (Biogen). At the indicated times the monolayers were washed three times with HBSS, and cells were scraped from the culture dishes into 0.5 ml of SPG solution and frozen until all samples were collected. Samples were briefly sonicated to disrupt the HeLa cells and release the chlamydial EBs. The disrupted cell suspensions were diluted in SPG solution and used to inoculate fresh monolayers of HeLa cells, as described above. Infected monolayers were cultured in MEM-10 containing cycloheximide at 1 ,ug/ml for 48 hr, then washed once with HBSS, and fixed with methanol; inclusions were visualized by indirect immunofluorescence with an anti-major outer membrane protein (MOMP) monoclonal antibody (mAb) (A-20) (14,15).In experiments to determine whether infectious chlamydiae could be res...

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Active inflammation and NF-κB activation contribute fundamentally to atherogenesis and plaque disruption. Accumulating evidence has implicated specific infectious agents including Chlamydia pneumoniae in the progression of atherogenesis. Chlamydial heat shock protein 60 (cHSP60) has been implicated in the induction of deleterious immune responses in human chlamydial infections and has been found to colocalize with infiltrating macrophages in atheroma lesions. cHSP60 might stimulate, enhance, and maintain innate immune and inflammatory responses and contribute to atherogenesis. In this study, we investigated the signaling mechanism of cHSP60. Recombinant cHSP60 rapidly activated NF-κB in human microvascular endothelial cells (EC) and in mouse macrophages, and induced human IL-8 promoter activity in EC. The inflammatory effect of cHSP60 was heat labile, thus excluding a role of contaminating LPS, and was blocked by specific anti-chlamydial HSP60 mAb. In human vascular EC which express Toll-like receptor 4 (TLR4) mRNA and protein, nonsignaling TLR4 constructs that act as dominant negative blocked cHSP60-mediated NF-κB activation. Furthermore, an anti-TLR4 Ab abolished cHSP60-induced cellular activation, whereas a control Ab had no effect. In 293 cells, cHSP60-mediated NF-κB activation required both TLR4 and MD2. A dominant-negative MyD88 construct also inhibited cHSP60-induced NF-κB activation. Collectively, our results indicate that cHSP60 is a potent inducer of vascular EC and macrophage inflammatory responses, which are very relevant to atherogenesis. The inflammatory effects are mediated through the innate immune receptor complex TLR4-MD2 and proceeds via the MyD88-dependent signaling pathway. These findings may help elucidate the mechanisms by which chronic asymptomatic chlamydial infection contribute to atherogenesis.

CD4؉ T-helper type 1 (Th1) responses are essential for the resolution of a primary Chlamydia trachomatis genital tract infection; however, elements of the immune response that function in resistance to reinfection are poorly understood. Defining the mechanisms of immune resistance to reinfection is important because the elements of protective adaptive immunity are distinguished by immunological memory and high-affinity antigen recognition, both of which are crucial to the development of efficacious vaccines. Using in vivo antibody depletion of CD4 ؉ and CD8 ؉ T cells prior to secondary intravaginal challenge, we identified lymphocyte populations that functioned in resistance to secondary chlamydial infection of the genital tract. Depletion of either CD4؉ or CD8 ؉ T cells in immune wild-type C57BL/6 mice had a limited effect on resistance to reinfection. However, depletion of CD4 ؉ T cells, but not CD8 ؉ T cells, in immune B-cell-deficient mice profoundly altered the course of secondary infection. CD4-depleted B-cell-deficient mice were unable to resolve a secondary infection, shed high levels of infectious chlamydiae, and did not resolve the infection until 3 to 4 weeks following the discontinuation of anti-CD4 treatment. These findings substantiated a predominant role for CD4؉ T cells in host resistance to chlamydial reinfection of the female genital tract and demonstrated that CD8 ؉ T cells are unnecessary for adaptive immune resistance. More importantly, however, this study establishes a previously unrecognized but very significant role for B cells in resistance to chlamydial reinfection and suggests that B cells and CD4 ؉