Purification of Chlamydia trachomatis strains in mixed infection by monoclonal antibody neutralization.

A Chlamydia trachomatis D* serovariant strain was found to be mixed with an F serovar strain in a clinical specimen. By using a monoclonal antibody which neutralizes serovar F infectivity in hamster kidney cells, the D* variant strain was enriched until it could be cloned by limiting dilution. This newly described neutralization enrichment procedure can be used to purify a C. trachomatis serovar present in small numbers in a mixed culture or, potentially, to identify nonneutralizable mutants.     

Monoclonal antibodies in serovar determination of 53 Chlamydia trachomatis isolates from Amiens, France

The serovar distribution of 53 Chlamydia trachomatis strains obtained from 53 clinical isolates in Amiens (France) was studied by a micro-immunofluorescence test with a panel of 15 monoclonal antibodies. The isolates were of ocular (babies) or urogenital origin (adults). This typing showed that E was the most common serovar (62.3%) followed by F (9.4%), Ba, D, J (5.6%), H (3.8%) and G, K (1.9%). Two mixed infections were detected (one EG and one FG). Consequently, the serovar distribution of C. trachomatis in Amiens (France), was characterized by a predominance of serovar E higher than in other European countries.     

Chlamydia trachomatis (L2 serovar) binds to distinct subpopulations of human peripheral blood leukocytes

We have previously shown that infants with pneumonitis caused by Chlamydia trachomatis, an obligate intracellular bacterium, possess increased percentages of B lymphocytes but not T lymphocytes in their peripheral blood. It was then demonstrated that chlamydiae induce proliferation in vitro of human peripheral blood B lymphocytes and, in the presence of T cells, differentiation of B cells to immunoglobulin-secreting cells. In this study, we show that C. trachomatis (L2 serovar) binds preferentially to 50% of human B lymphocytes from peripheral blood but only to a small percentage, if any, of T cells. Both monocytes and granulocytes bind and ingest chlamydiae. Despite chlamydial binding to B cells and ingestion by monocytes, no uptake by B cells and limited growth (fewer than 0.5% inclusion-containing cells) in monocytes occur. There is a dramatic decrease in the percentage of cells associated with the bacteria after culture. These results are the first demonstration of binding of C. trachomatis (L2 serovar) to lymphocytes and represent a direct step toward correlating physical interactions between bacteria and lymphocytes with specific immunostimulatory activities in vitro.     

Complement dependent in vitro neutralization of Chlamydia trachomatis by a subspecies-specific monoclonal antibody

Three murine monoclonal antibodies which combined specifically with Chlamydia trachomatis strain HAR-13 (Serotype A) were characterized. All antibodies cross-reacted with subspecies-specific epitopes in previously undescribed patterns. One of the antibodies showed a complement-dependent partial neutralization of strain HAR-13 in vitro, whereas the remaining two antibodies did not neutralize under any experimental conditions tested. The significance of subspecies-specific antibodies in neutralizing chlamydial infectivity in vivo remains to be determined.     

Ocular sensitization of mice by live (but not irradiated) Chlamydia trachomatis serovar A.

Ocular exposure of mice to live elementary bodies of Chlamydia trachomatis serovar A results in immunological sensitization of the mice. This reactivity is manifested by the development of early (5 h) and delayed-type (24 h) dermal reactivity and serovar-specific antibody formation against either live or irradiated (100 kilorads) elementary bodies. Parallel ocular exposure of mice to irradiated elementary bodies does not result in this sensitization. The early and late dermal immune responses induced by ocular exposure to live organisms can be transferred to unexposed mice by serum and lymphoid cell transfers, respectively. It appears that successful murine ocular sensitization by human C. trachomatis serovar A elementary bodies is an ability manifested by live organisms and not by inactivated but antigenic organisms.     

Co-incubation of human spermatozoa with Chlamydia trachomatis serovar E causes premature sperm death

The aim of this work was to investigate the effect of elementary bodies (EB) of Chlamydia trachomatis serovars E and LGV on sperm motility, viability and acrosomal status. Highly motile preparations of spermatozoa from normozoospermic patients were co-incubated for 6 h with 0.54x10(6) EB per ml. At 1, 3 and 6 h of incubation, sperm motility was determined by computer-assisted semen analysis (CASA) and the proportion of dead cells determined by the hypo-osmotic swelling (HOS) test. Acrosomal status was also examined using a standard monoclonal antibody assay. In the absence of EB, the percentage of motile spermatozoa remained >69% over the 6h incubation and the proportion of dead spermatozoa at <12%. However, during the incubation with EB of serovar E there was a significant decline in the percentage of motile spermatozoa (P < 0.05), and a corresponding increase in the proportion of dead spermatozoa (P < 0.05) at all time-points. However, following incubation with serovar LGV, only the percentage of dead spermatozoa after 6 h incubation was significantly different from the control (P < 0.05). The amount of acrosome-reacted spermatozoa remained unchanged (<16%) in all incubations at all time-points. Dose-response experiments indicated that increasing the concentration of EB to 2.5x10(6) per ml did not significantly alter the results. Furthermore, co-incubation of spermatozoa with dead EB (killed by heat treatment) abolished the chlamydia-mediated response, indicating that the effect is a result of the live organism and not soluble components or membrane elements. These data suggest that a detrimental effect on sperm function by some serovars may be an as yet unrecognized component of infertility problems.     

Early complement components enhance neutralization of Chlamydia trachomatis infectivity by human sera.

Immunoglobulin G in human serum neutralizes chlamydial infectivity in vitro. Complement-intact, C5-depleted, and C8-depleted human serum all have significantly more neutralizing activity than serum heated to inactivate early components of complement. Cobra venom factor, an analog of human C3b, enhances neutralization of antichlamydial immunoglobulin G in the absence of early complement components.     

Effects of antibody isotype and host cell type on in vitro neutralization of Chlamydia trachomatis.

Monoclonal antibodies (MAbs) E-4, E-21, and DIII A3, which recognize the same or similar overlapping peptides in the variable domain IV of the major outer membrane protein of Chlamydia trachomatis but differ in isotype, were used in a complement-independent (CI) in vitro neutralization assay. These MAbs had previously been shown to neutralize chlamydial infectivity in HeLa 229 cells in a complement-dependent assay. In this report, all three MAbs neutralized chlamydial infectivity in HaK cells in a CI assay. However, when HeLa cells were used as the host cell, MAb E-4 (immunoglobulin G2b [IgG2b]) and MAb DIII A3 (IgG2b) failed to neutralize infectivity, while MAb E-21 (IgG1) neutralized chlamydial infectivity. These findings are consistent with the proposal that because of the presence of Fc gamma RIII receptors, HeLa cells facilitate infectivity and thus block neutralization through the uptake of an IgG2b-chlamydia complex. Since Fc gamma RIII receptors do not bind or bind poorly to IgG1, neutralization of C. trachomatis by MAb E-21 in HeLa cells is also corroborative evidence for the role of Fc gamma RIII receptors in this interaction. A fivefold enhancement of infectivity was seen when 10 and 1 micrograms of MAb E-4 per ml were tested in a CI assay with HeLa cells. In performing CI neutralization synergy studies in HeLa cells with MAbs E-4 and E-21, antagonism between MAbs E-4 and E-21 was observed at MAb E-4 concentrations of 10 and 1 micrograms/ml for all concentrations of MAb E-21 tested (10 to 0.1 micrograms/ml). When HaK cells were used in the same studies, no antagonism between the MAbs was found. In addition, when HeLa cells were used in a CI assay, polyclonal serum raised to a peptide representing variable domain IV of the major outer membrane protein inhibited the neutralizing ability of MAb E-21. The blocking of neutralization and the enhancement of infectivity by chlamydia-specific antibodies seen in this investigation with HeLa cells may have important clinical implications for developing preventive strategies for chlamydial infections.     

Differential regulation of inflammatory cytokine secretion by human dendritic cells upon Chlamydia trachomatis infection

Chlamydia trachomatis is an obligate intracellular gram-negative bacterium responsible for a wide spectrum of diseases in humans. Both genital and ocular C. trachomatis infections are associated with tissue inflammation and pathology. Dendritic cells (DC) play an important role in both innate and adaptive immune responses to microbial pathogens and are a source of inflammatory cytokines. To determine the potential contribution of DC to the inflammatory process, human DC were infected with C. trachomatis serovar E or L2. Both C. trachomatis serovars were found to infect and replicate in DC. Upon infection, DC up-regulated the expression of costimulatory (B7-1) and cell adhesion (ICAM-1) molecules. Furthermore, chlamydial infection induced the secretion of interleukin-1beta (IL-1beta), IL-6, IL-8, IL-12p70, IL-18, and tumor necrosis factor alpha (TNF-alpha). The mechanisms involved in Chlamydia-induced IL-1beta and IL-18 secretion differed from those of the other cytokines. Chlamydia-induced IL-1beta and IL-18 secretion required infection with viable bacteria and was associated with the Chlamydia-induced activation of caspase-1 in infected host cells. In contrast, TNF-alpha and IL-6 secretion did not require that the Chlamydia be viable, suggesting that there are at least two mechanisms involved in the Chlamydia-induced cytokine secretion in DC. Interestingly, an antibody to Toll-like receptor 4 inhibited Chlamydia-induced IL-1beta, IL-6, and TNF-alpha secretion. The data herein demonstrate that DC can be infected by human C. trachomatis serovars and that chlamydial components regulate the secretion of various cytokines in DC. Collectively, these data suggest that DC play a role in the inflammatory processes caused by chlamydial infections.     

Differences in innate immune responses (in vitro) to HeLa cells infected with nondisseminating serovar E and disseminating serovar L2 of Chlamydia trachomatis

The inflammatory response associated with Chlamydia trachomatis genital infections is thought to be initiated by the release of proinflammatory cytokines from infected epithelial cells. This study focuses on the interactions between C. trachomatis-infected HeLa cells and immune cells involved in the early stages of infection, i.e., neutrophils and macrophages. First, we showed that the expression of interleukin-11 (IL-11), an anti-inflammatory cytokine mainly active on macrophages, was upregulated at the mRNA level in the genital tracts of infected mice. Second, incubation of differentiated THP-1 (dTHP-1) cells or monocyte-derived macrophages (MdM) with basal culture supernatants from C. trachomatis serovar E- or serovar L2-infected HeLa cells resulted in macrophage activation with a differential release of tumor necrosis factor alpha (TNF-alpha) and upregulation of indoleamine 2,3-deoxygenase (IDO) but not of Toll-like receptor 2 and 4 mRNA expression. Third, coculture of infected HeLa cells with dTHP-1 cells resulted in a reduction in chlamydial growth, which was more dramatic for serovar E than for L2 and which was partially reversed by the addition of anti-TNF-alpha antibodies for serovar E or exogenous tryptophan for both serovars but was not reversed by the addition of superoxide dismutase or anti-IL-8 or anti-IL-1beta antibodies. A gamma interferon-independent IDO mRNA upregulation was also detected in dTHP-1 cells from infected cocultures. Lastly, with a two-stage coculture system, we found that (i) supernatants from neutrophils added to the apical side of infected HeLa cell cultures were chlamydicidal and induced MdM to express antichlamydial activity and (ii) although polymorphonuclear leukocytes released more proinflammatory cytokines in response to serovar E- than in response to L2-infected cells, MdM were strongly activated by serovar L2 infection, indicating that the early inflammatory response generated with a nondisseminating or a disseminating strain is different.     

Binding, ingestion, and multiplication of Chlamydia trachomatis (L2 serovar) in human leukocyte cell lines.

We examined the ability of lymphoblastoid-myeloid cell lines to bind, ingest, and permit multiplication of Chlamydia trachomatis (L2 serovar). Four types of chlamydia-cell line interactions were observed: minimal bacterial binding; bacterial binding, followed by ingestion and high-level multiplication; bacterial binding, followed by ingestion but minimal multiplication; and bacterial binding, but minimal ingestion or replication. Our data demonstrate that at 37 degrees C in vitro the L2 serovar can both bind avidly to a cell without entering it and enter nonphagocytic cells but not grow.     

Cytoskeletal requirements in Chlamydia trachomatis infection of host cells.

Infection of genital epithelial cells by the closely related sexually transmitted pathogens Chlamydia trachomatis serovars E and L2 results in different clinical disease manifestations. Following entry into target host cells, individual vesicles containing chlamydiae fuse with one another to form one large inclusion. At the cellular level, the only obvious difference between these serovars is the time until inclusion maturation, which is 48 h for the invasive serovar L2 and 72 h for serovar E. To begin to define the intracellular events of these pathogens, the effect of cytoskeletal disruption on early endosome fusion and inclusion development in epithelial (HEC-1B) and fibroblast (McCoy) cells was analyzed by fluorescence microscopy. Disruption of microfilaments with cytochalasin D markedly reduced serovar E, but not serovar L2, infection of both cell lines. Conversely, microfilament as well as microtubule disruption, with colchicine or nocodazole, had no effect on serovar E inclusion development but resulted in the formation of multiple serovar L2 inclusions per cell during early and mid-development. Later in serovar L2 inclusion development (> 36 h postinfection), vesicles containing chlamydiae fused to form one large inclusion in the absence of an intact cytoskeleton. These results imply that (i) C. trachomatis serovar E may utilize a different pathway for uptake and development from serovar L2; (ii) these differences are consistent in both epithelial cells and fibroblasts; and (iii) the cytoskeleton plays a unique role in the infection of host cells by these two genital pathogens.     

Persistent infection of mouse fibroblasts (McCoy cells) with a trachoma strain of Chlamydia trachomatis.

An in vitro model of persistent infection of mouse fibroblasts (McCoy cells) with a trachoma strain (G17) of Chlamydia trachomatis has been developed. Persistently infected cultures were established by infecting McCoy cells with high multiplicities of chlamydiae. After the first cycle of chlamydial replication, the host cells multiplied more rapidly than the parasites, so that the fraction of inclusion-bearing cells declined to less than 1%. However, after 100 days, the proportion of inclusion-bearing cells rose dramatically, and the cultures alternated between periods of massive host cell destruction by chlamydiae and periods of host cell proliferation. This cycle continued indefinitely as host cell and parasite densities fluctuated periodically. The chlamydiae in the cycling populations were reidentified as the original serotype. No changes in either host cell susceptibility or chlamydial invasiveness were observed in hosts and parasites recovered from persistently infected populations. All evidence suggests that the parasite maintained itself in McCoy cell populations by cell-to-cell transfer and that an equilibrium between host and parasite multiplication was achieved when the persistently infected cultures fluctuated between periods of host cell destruction and proliferation.     

Chlamydial Hsp60-2 is iron responsive in Chlamydia trachomatis serovar E-infected human endometrial epithelial cells in vitro

Chlamydial 60-kDa heat shock proteins (cHsp60s) are known to play a prominent role in the immunopathogenesis of disease. It is also known that several stress-inducing growth conditions, such as heat, iron deprivation, or exposure to gamma interferon, result in the development of persistent chlamydial forms that often exhibit enhanced expression of cHsp60. We have shown previously that the expression of cHsp60 is greatly enhanced in Chlamydia trachomatis serovar E propagated in an iron-deficient medium. The objective of this work was to determine which single cHsp60 or combination of the three cHsp60 homologs encoded by this organism responds to iron limitation. Using monospecific polyclonal peptide antisera that recognize only cHsp60-1, cHsp60-2, or cHsp60-3, we found that expression of cHsp60-2 is responsive to iron deprivation. Overall, our studies suggest that the expression of cHsp60 homologs differs among the mechanisms currently known to induce persistence.     

Sulfated polyanions block Chlamydia trachomatis infection of cervix-derived human epithelia.

Using a cell line derived from the human cervix and a rapid fluorescence cytotoxicity assay, we have shown that Chlamydia trachomatis infection can be blocked by certain sulfated polysaccharides (carrageenan, pentosan polysulfate, fucoidan, and dextran sulfate) and glycosaminoglycans (heparin, heparan sulfate, and dermatan sulfate) but not by other glycosaminoglycans (chondroitin sulfate A or C, keratan sulfate, and hyaluronic acid). The most negatively charged molecules are the most effective at blocking infection. Results of infection at 4 degrees C suggest that sulfated polyanions act by preventing the adherence of chlamydiae to target cells. These and additional blocking studies with enzymes suggest that a heparan sulfate-like glycosaminoglycan on the surface of elementary bodies is involved in the adherence of chlamydiae to target cells, probably through a nonspecific charge interaction or possibly a heparin-binding protein. We previously observed that the same sulfated polysaccharides inhibit transmission of human immunodeficiency virus in vitro and suggested that these compounds could be used in a vaginal formulation to inhibit infection by human immunodeficiency virus. The results of the present study suggest that the same type of formulation may inhibit sexual transmission of chlamydia.     

Role of gamma-delta T cells in murine Chlamydia trachomatis infection.

The role of gamma-delta T cells in host resistance to Chlamydia trachomatis was characterized by using a murine model of pneumonia caused by the mouse pneumonitis agent (MoPn), murine C. trachomatis. At days 3 and 7 after infection, gamma-delta T-cell-deficient knockout mice had significantly higher levels of MoPn in the lungs than did immunologically intact controls. At day 20, paradoxically, gamma-delta T-cell-deficient mice were more resistant to MoPn than were controls. This increased resistance was not due to an increased production of toxic cytokines or interleukin-10 in controls on that day. Gamma-delta T cells play a role in protection early in MoPn infection, but they may be deleterious later in infection, as has been observed in models of salmonella and trypanosome infection.     

Differences in cell activation by Chlamydophila pneumoniae and Chlamydia trachomatis infection in human endothelial cells

Seroepidemiological studies and demonstration of viable bacteria in atherosclerotic plaques have linked Chlamydophila pneumoniae infection to the development of chronic vascular lesions and coronary heart disease. In this study, we characterized C. pneumoniae-mediated effects on human endothelial cells and demonstrated enhanced phosphorylation and activation of the endothelial mitogen-activated protein kinase (MAPK) family members extracellular receptor kinase (ERK1/2), p38-MAPK, and c-Jun-NH2 kinase (JNK). Subsequent interleukin-8 (IL-8) expression was dependent on p38-MAPK and ERK1/2 activation as demonstrated by preincubation of endothelial cells with specific inhibitors for the p38-MAPK (SB202190) or ERK (U0126) pathway. Inhibition of either MAPK had almost no effect on intercellular cell adhesion molecule 1 (ICAM-1) expression. While Chlamydia trachomatis was also able to infect endothelial cells, it did not induce the expression of endothelial IL-8 or ICAM-1. These effects were specific for a direct stimulation with viable C. pneumoniae and independent of paracrine release of endothelial cell-derived mediators like platelet-activating factor, NO, prostaglandins, or leukotrienes. Thus, C. pneumoniae triggers an early signal transduction cascade in target cells that could lead to endothelial cell activation, inflammation, and thrombosis, which in turn may result in or promote atherosclerosis.     

Uptake and processing of Chlamydia trachomatis by human dendritic cells

Chlamydia trachomatis (CT) causes several sexually transmitted diseases. In 2-5% of cases, CT infection leads to the development of reactive arthritis. Dendritic cells (DC) are central in T cell priming and the induction of antigen specific immunity. Here we have studied the uptake and processing of CT serovar L2 by human DC, and their ability to present CT antigens to both CD4(+) and CD8(+) T cells. We show that the entry of CT was mediated by the attachment of CT to heparan sulfates and could be inhibited by heparin. There was no inhibition of uptake by an agent which blocks micropinocytosis. Infecting DC with CT led to their activation and the production of IL-12 and TNF-alpha but not IL-10. Following invasion, CT was confined to distinct vacuoles which were visualized with anti-CT antibodies using confocal microscopy. Unlike with epithelial cells, these vacuoles did not develop into characteristic inclusion bodies. In the first 48 h, CT(+) vacuoles were negative for Lamp-1 and MHC class II. Despite no obvious co-localization between CT vacuoles and MHC loading compartments, infected DC efficiently presented CT antigens to CD4(+) T cells. Infected DC also expanded CT specific CD8(+) T cells, allowing us to generate a number of CT-reactive CD8(+) T cell clones. There is still controversy about the importance of chlamydia-specific CD8(+) T cell responses in patients with arthritis. This is largely due to the difficulties in studying CTL responses at the clonal level. The use of DC as antigen-presenting cells should enable more detailed characterization of these CTL responses.     

In vitro neutralization of Chlamydia trachomatis by monovalent Fab antibody specific to the major outer membrane protein.

Monovalent Fab antibodies to serovar- and subspecies-specific epitopes of the major outer membrane protein (MOMP) of Chlamydia trachomatis neutralized infectivity for hamster kidney cells by preventing chlamydial attachment. These findings exclude the aggregation of chlamydiae as a mechanism of anti-MOMP neutralization and provide additional evidence in support of the MOMP as a chlamydial adhesin.