Morphologic and antigenic characterization of interferon gamma-mediated persistent Chlamydia trachomatis infection in vitro.

Abstract

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...

Effect of the steroid receptor antagonist RU486 (mifepristone) on an IFNγ-induced persistent Chlamydophila pneumoniae infection model in epithelial HEp-2 cells

“…Gene expression of the 16S rRNA, which is critical for C. pneumoniae survival in cells, remained unchanged. Thus, persistent C. pneumoniae infection in HEp-2 cells was stably induced using IFN and supported the persistent infection models described previously [26,28].…”

“…Persistent infection of C. pneumoniae in HEp-2 cells was induced by the treatment with IFN, since accumulating evidence from studies using in vitro persistent infection models suggested that IFN was the most effective reagent for inducing persistent C. pneumoniae infection with an incomplete developmental cycle [1,26,28]. As a result, treatment with IFN had an obvious effect on the growth of C. pneumoniae, resulting in a decrease in number of infectious progenies and a reduction of inclusion size.…”

“…Previous works suggested that persistent chlamydial infection might be associated with incomplete chlamydial development with sporadic production of EBs refractory to treatment with antibiotics, although its reason remains unknown [26,28]. Therefore, we assumed that a decrease of the sporadic EBs with a drug might be a significant indicator to evaluate the elimination of C. pneumoniae in persistent infection, and assessed changes of the numbers of infectious progenies (by IFU assay) in persistent infection.…”

“…The persistent form of C. pneumoniae in human epithelial HEp-2 cells was induced by treatment with IFN according to the method described previously [28].…”

Pathogenesis of spondylarthropathies

“…We have shown recently that IL-4 was more often present in ReA patients compared with rheumatoid arthritis patients, possibly mediating bacterial persistence in the joint by inhibition of IFNy effects (73). The importance of an optimal IFNy concentration for effective elimination of Chlamydia and Yersinia has recently been demonstrated in vitro (41) and in an animal model (74,75). Therefore, in those cases of ReA and spondylarthropathy in which bacterial antigen is present in the joint, the presence of IL-4 may indicate an ineffective immune response.…”