Chlamydia trachomatis and chlamydial heat shock protein 60-specific antibody and cell-mediated responses predict tubal factor infertility.

BACKGROUND: To evaluate the role of Chlamydia trachomatis-induced humoral and cell-mediated immune (CMI) responses in predicting tubal factor infertility (TFI). METHODS: Blood samples were taken from 88 women with TFI and 163 control women. C. trachomatis and chlamydial heat shock protein 60 (CHSP60)-specific immunoglobulin G (IgG) antibodies were analysed using enzyme-linked immunosorbent assay (ELISA) kits. Proliferative reactivity of peripheral blood mononuclear cells was studied in vitro against Chlamydia elementary body (EB) and recombinant CHSP60 antigens. RESULTS: C. trachomatis-specific IgG antibodies were found more frequently (43.2 versus 13.5%), and the antibody levels were higher in the TFI cases than in the controls (P < 0.001). C. trachomatis EB-induced lymphocyte responses were positive in 81.8% of the TFI cases and 58.9% of the controls (P < 0.001). Similarly, CHSP60-induced lymphocyte responses were found in 45.5% of the TFI cases and 30.7% of the controls (P < 0.001). CHSP60 antibody test was the best single test predicting TFI. Compared to cases with all four markers negative, the estimated risk for TFI was 4.1 (95% CI 1.4-11.9) among those with one positive marker and 19.9 (95% CI 6.9-57.4) among those with three to four positive markers. CONCLUSION: Our results show that TFI prediction model can be improved by combining tests for humoral and CMI response to chlamydial antigens.

Chlamydia trachomatis heat shock protein-60 induced interferon-gamma and interleukin-10 production in infertile women.

Chlamydia trachomatis-associated tubal factor infertility (TFI) involves enhanced humoral and cell-mediated immune response to the chlamydial 60 kDa heat shock protein (CHSP60). We evaluated the role of CHSP60-induced immune response in TFI by studying lymphocyte proliferation and cytokine (interferon (IFN)-gamma, interleukin (IL)-12 and IL-10) secretion in response to C. trachomatis elementary body (EB) and CHSP60 antigens in 57 women with TFI and in 76 women with other causes of infertility. Positive proliferative response of PBMC to CHSP60 was more common in the TFI group (20/57; 36%) than in the other groups (17/76; 22%) although the frequency or the median responses did not differ significantly (1.6, range 0.2-22.1 versus 1.4; 0.2-24.4). C. trachomatis EB induced significantly higher IFN-gamma and lower IL-10 secretion in the TFI group compared to the other groups. The EB and CHSP60 induced IL-12 secretion was similar in all study groups and correlated with IFN-gamma secretion in the other but not in the TFI group. The lack of correlation between EB-induced IL-12 and IFN-gamma production and simultaneously found prominent IL-10 secretion in response to CHSP60 in the TFI group suggests that the CHSP60 may have a specific role in regulating the immune reactions during chlamydial infection and may consequently contribute to the immunopathogenesis of TFI.

QSYP peptide sequence is selected from phage display libraries by bovine IgG contaminants in monoclonal antibody preparations.

A consensus peptide sequence, QSYP, appears as an artifact during the mapping of monoclonal antibodies (MAbs) using a random peptide phage display library. Phage bearing this QSYP sequence were independently selected by four different laboratories screening separate MAb preparations with the same phage library. In each case, the QSYP sequence was selected in addition to a consensus sequence specific to the MAb. Phage that displayed the QSYP sequence were not bound by the MAb of interest, but rather bound to bovine IgG derived from the FBS present in the hybridoma growth media. The implications of this finding for the interpretation of phage library screening results and possible methods for the removal of bovine IgG from MAb preparations are discussed.

HLA DQ alleles and interleukin-10 polymorphism associated with Chlamydia trachomatis-related tubal factor infertility: a case-control study.

BACKGROUND: The relationship between Chlamydia trachomatis tubal factor infertility (TFI) and the host's immunoregulatory genes was studied. METHODS: Cell-mediated immune responses to C. trachomatis and chlamydial heat shock protein (CHSP60) were determined by lymphocyte proliferation assay. HLA-DQ alleles and interleukin-10 (IL-10) promoter polymorphism (-1082 A/G) were analysed in 52 TFI cases and in 61 controls by PCR. RESULTS: HLA-DQB1 or DQA1 alleles did not significantly differ between the TFI group and the control group. However, DQA1*0102 and DQB1*0602 alleles together with IL-10 -1082AA genotype were found significantly more frequently in the TFI patients than in the controls (0.18 and 0.02 respectively; P = 0.005). Five (22%) of the 23 patients who had a positive lymphocyte proliferative response to CHSP60 were positive also for IL-10 -1082AA and for the HLA-DQA1*0102 and HLA-DQB1*0602 alleles. CONCLUSIONS: Our results reveal an association of a cellular immune response to CHSP60, HLA class II alleles and IL-10 promoter genotypes in patients with chlamydial TFI.

Resolution of secondary Chlamydia trachomatis genital tract infection in immune mice with depletion of both CD4+ and CD8+ T cells.

The essential role of T cells in the resolution of primary murine Chlamydia trachomatis genital tract infection is inarguable; however, much less is known about the mechanisms that confer resistance to reinfection. We previously established that CD4+ T cells and B cells contribute importantly to resistance to reinfection. In our current studies, we demonstrate that immune mice concurrently depleted of both CD4+ T cells and CD8+ T cells resisted reinfection as well as immunocompetent wild-type mice. The in vivo depletion of CD4+ and CD8+ T cells resulted in diminished chlamydia-specific delayed-type hypersensitivity responses, but antichlamydial antibody responses were unaffected. Our data indicate that immunity to chlamydial genital tract reinfection does not rely solely upon immune CD4+ or CD8+ T cells and further substantiate a predominant role for additional effector immune responses, such as B cells, in resistance to chlamydial genital tract reinfection.

Immunity to murine Chlamydia trachomatis genital tract reinfection involves B cells and CD4(+) T cells but not CD8(+) T cells.

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(+) T cells may function synergistically in providing immunity in this model of chlamydial infection. Whether CD4(+) T cells and B cells function independently or dependently is unknown, but definition of those mechanisms is fundamental to understanding optimum protective immunity and to the development of highly efficacious immunotherapies against chlamydial urogenital infections.

Differential sensitivities of Chlamydia trachomatis strains to inhibitory effects of gamma interferon.

Gamma interferon (IFN-gamma) is an important cytokine in host defense against chlamydial infection. An in vitro cell culture system was used to show that IFN-gamma inhibition of chlamydial growth, as determined by diminished recovery of infectious elementary bodies, differed markedly among chlamydial strains. These differences in sensitivity among chlamydial strains to IFN-gamma-mediated inhibition may profoundly influence the clinical outcome of infection.

Chlamydial virulence determinants in atherogenesis: the role of chlamydial lipopolysaccharide and heat shock protein 60 in macrophage-lipoprotein interactions.

Data from a spectrum of epidemiologic, pathologic, and animal model studies show that Chlamydia pneumoniae infection is associated with coronary artery disease, but it is not clear how the organism may initiate or promote atherosclerosis. It is postulated that C. pneumoniae triggers key atherogenic events through specific virulence determinants. C. pneumoniae induces mononuclear phagocyte foam cell formation by chlamydial lipopolysaccharide (cLPS) and low-density lipoprotein oxidation by chlamydial hsp60 (chsp60). Thus, different chlamydial components may promote distinct events implicated in the development of atherosclerosis. Data implicating cLPS and chsp60 in the pathogenesis of atherosclerosis are discussed and novel approaches are presented for attempting to elucidate how these putative virulence determinants signal mononuclear phagocytes to modulate lipoprotein influx and modification.

In situ analysis of the evolution of the primary immune response in murine Chlamydia trachomatis genital tract infection.

Adaptive immune responses contribute to the resolution of Chlamydia trachomatis genital tract infection and protect against reinfection, but our understanding of the mechanisms of those protective responses is incomplete. In this study, we analyzed by in situ immunohistochemistry the progression of the inflammatory and cytokine responses in the genital tracts of mice vaginally infected with C. trachomatis strain mouse pneumonitis. The cellular inflammatory response was characterized by an initial elevation in myeloid cells in the vagina (day 3) and uterine horns (day 7), followed by a marked rise in the number of T cells, predominantly CD4(+) cells. CD8(+) T cells and CD45R(+) B cells were also detected but were much less numerous. Perivascular clusters of CD4(+) T cells, which resembled clusters of T cells seen in delayed-type hypersensitivity responses, were evident by 2 weeks postinfection. Following the resolution of infection, few CD8(+) T cells and CD45R(+) B cells remained, whereas numerous CD4(+) T cells and perivascular clusters of CD4(+) T cells persisted in genital tract tissues. Interleukin-12 (IL-12)- and tumor necrosis factor alpha (TNF-alpha)-producing cells were observed in vaginal tissue by day 3 of infection and in uterine tissues by day 7. Cells producing IL-4 or IL-10 were absent from vaginal tissues at day 3 of infection but were present in uterine tissues by day 7 and were consistently more numerous than IL-12- and TNF-alpha-producing cells. Thus, the evolution of the local inflammatory response was characterized by the accumulation of CD4(+) T cells into perivascular clusters and the presence of cells secreting both Th1- and Th2-type cytokines. The persistence of CD4(+)-T-cell clusters long after infection had resolved (day 70) may provide for a readily mobilizable T-cell response by which previously infected animals can quickly respond to and control a secondary infectious challenge.

Detection of Chlamydia pneumoniae-reactive T lymphocytes in human atherosclerotic plaques of carotid artery.

Linkage between Chlamydia pneumoniae infection and atherosclerosis has been confirmed in several studies, but the precise role of this organism in the disease process is not known. We investigated the relation and reactivity of T lymphocytes of human carotid plaques to C pneumoniae antigens. Tissue specimens were obtained from 17 patients who underwent carotid endarterectomy. Immunohistological staining and/or in situ hybridization revealed the presence of C pneumoniae in 11 (64%) of the 17 of the cases. Inflammatory infiltration seen in the vessel walls consisted primarily of CD45RO+ T-memory lymphocytes (median 80%, range 50% to 90%), whereas CD20+ B cells and monocytes were in minor proportion. In vivo activated T lymphocytes were propagated from the specimens with interleukin-2, and the antigen specificity of the established T-cell lines (TLLs) was analyzed against C pneumoniae elementary body antigen. TLLs were established from all 17 carotid tissues but none from the control specimens of ascending aorta. C pneumoniae was recognized as a specific T-cell-stimulating antigen in 7 (41%) of 17 cases. Further analyses of the C pneumoniae-reactive TLLs showed that chlamydial 60-kDa heat-shock protein induced specific proliferation in 5 (71%) of 7 cases and revealed 2 haplotype (DRB1*1502 and DQB1*06) binding motifs in human 60-kDa heat-shock protein. C pneumoniae was identified as a specific microbial antigen recognized by 41% of TLLs propagated from in vivo activated plaque T cells. Our results suggests that cell-mediated immunity to C pneumoniae plays a role in the atherosclerotic process and that this response may involve autoimmunity.

Seroreactivity to Chlamydia trachomatis Hsp10 correlates with severity of human genital tract disease.

We have identified the chlamydial heat shock protein Hsp10 as a potential correlate to the immunopathogenic process in women with tubal factor infertility (TFI). The human serologic response to chlamydial Hsp10, Hsp60, and major outer membrane protein (MOMP) was measured by enzyme-linked immunosorbent assay. Three populations of women were studied: uninfected controls (CU), acutely infected (AI) women, and women with TFI. Sera from women in the AI and TFI groups both recognized Hsp10 more frequently and at a higher overall level than sera from healthy uninfected controls. Moreover, the infertile women had significantly greater Hsp10 seroreactivity than acutely infected women, indicating a concomitant increase of Hsp10 recognition in populations with increasing levels of disease severity. Hsp60 reactivity showed a similar correlation in these populations, while MOMP reactivity peaked at the same level in both AI and TFI populations but did not increase with disease severity. Test populations were standardized by level of reactivity to formalin-fixed Chlamydia trachomatis elementary bodies (EBs) to address whether these associations were reflections of increased overall chlamydial exposure rather than a property specific to Hsp10. Associations between Hsp10 seropositivity and TFI were greater in the EB(+) subgroup while associations among the EB(-) subgroup were diminished. When restricted to the EB(+) subgroups, Hsp60 and MOMP responses in the TFI population did not increase significantly over the level of AI group responses. Thus, among women with similar exposure to chlamydiae, the serologic response to Hsp10 exhibited a stronger correlation with TFI than did the response to Hsp60 or MOMP. These findings support the hypothesis that the serological response to C. trachomatis heat shock proteins is associated with the severity of disease and identifies Hsp10 as an antigen recognized by a significant proportion of women with TFI.

Cellular oxidation of low-density lipoprotein by Chlamydia pneumoniae.

A spectrum of clinical and epidemiologic studies implicate infectious agents, including Chlamydia pneumoniae, in the pathogenesis of atherosclerosis. The complexity of atherosclerotic disease necessitates examining the role of infection in the context of defined risk factors, such as high levels of native low-density lipoprotein (LDL). Although native LDL does not have atherogenic properties, cellular oxidation of LDL alters the lipoprotein into a highly atherogenic form. In this report, C. pneumoniae and chlamydial hsp60, an inflammatory antigen that was recently localized to atheromas, were found to induce cellular oxidation of LDL. These data provide initial evidence that an infectious agent can render LDL atherogenic and suggest a mechanism whereby C. pneumoniae may promote atheroma development.

A vaccine and monoclonal antibodies that enhance mouse resistance to Candida albicans vaginal infection.

We previously reported that a vaccine composed of liposome-mannan complexes of Candida albicans (L-mann) stimulates mice to produce protective antibodies against disseminated candidiasis. An immunoglobulin M (IgM) monoclonal antibody (MAb), B6.1, specific for a beta-1,2-mannotriose in the complexes protects against the disease, whereas MAb B6 does not. In the present study, the vaccine and MAbs B6.1 and B6 were tested for the ability to protect against Candida vaginal infection, established by intravaginal (i.vg.) inoculation of yeast cells in mice maintained in pseudoestrus. Fungal CFU in each vagina was determined to assess the severity of infection. Mice vaccinated before infection developed about 62% fewer vaginal CFU than nonimmunized controls. Naive mice that received polyclonal antiserum (from vaccinated mice) i.vg. before infection had 60% fewer CFU than controls. The serum protective factor was stable at 56 degreesC, but C. albicans cells absorbed this factor. Mice given MAb B6.1 i.vg. after infection was established had fewer Candida CFU in vaginal tissue than control mice given buffer instead of antibody. MAbs B6.1 and B6 given intraperitoneally before infection protected mice, but MAbs preabsorbed with yeast cells did not. MAb B6.1 also protected against C. tropicalis vaginal infection, but MAb B6 did not. The protective activities of MAbs B6.1 and B6 appeared to be specific because an irrelevant IgM carbohydrate-specific MAb and an irrelevant IgG protein-specific MAb were not protective; also, MAb B6.1 did not affect development of vaginal chlamydial infection. These studies show that an appropriate antibody response, or administration of protective antibodies, can help the host to resist Candida vaginal infection.

Chlamydia trachomatis genital tract infection of antibody-deficient gene knockout mice.

The importance of antibody-mediated immunity in primary and secondary Chlamydia trachomatis genital tract infections was examined by using a definitive model of B-cell deficiency, the microMT/microMT gene knockout mouse. Vaginally infected B-cell-deficient microMT/microMT mice developed a self-limiting primary infection that was indistinguishable from infection of control C57BL/6 mice. Sera and vaginal secretions from infected mice were analyzed for anti-Chlamydia antibodies. C57BL/6 mice produced high-titered serum anti-Chlamydia immunoglobulin G2a (IgG2a), IgG2b, and IgA antibodies, and vaginal washes contained predominately anti-Chlamydia IgA. Serum and vaginal washes from infected B-cell-deficient mice were negative for anti-Chlamydia antibody. T-cell proliferation and delayed-type hypersensitivity assays were used as measures of Chlamydia-specific cell-mediated immunity and were found to be comparable for C57BL/6 and B-cell-deficient mice. Seventy days following primary infection, mice were rechallenged to assess acquired immunity. B-cell-deficient mice which lack anti-Chlamydia antibodies were more susceptible to reinfection than immunocompetent C57BL/6 mice. However, acquired immune resistance was evident in both strains of mice and characterized by decreased shedding of chlamydiae and an infection of shorter duration. Thus, this study demonstrates that cell-mediated immune responses alone were capable of resolving chlamydial infection; however, in the absence of specific antibody, mice were more susceptible to reinfection. Therefore, these data suggest that both humoral and cell-mediated immune responses were important mediators of immune protection in this model, though cell-mediated immune responses appear to play a more dominant role.

Immune Protection Against Chlamydia trachomatis in Females.

Despite significant advances in our understanding of the biology and antigenic structure of Chlamydia trachomatis, and the epidemiology and clinical spectrum of chlamydial disease, the magnitude of morbidity from human chlamydial infections remains an important public health concern. Control of chlamydial disease will likely depend on a multidisciplinary approach, including the development of immunoprophylactic or immunotherapeutic strategies. Reasonable progress has been made in understanding specific immune mechanisms that contribute to host immunity in experimental models of chlamydial infection. However, studies of human immunity have not been so successful. This is particularly evident in that studies to address the development and role of mucosal immune responses to urogenital chlamydial infections have not been forthcoming. The following review is a brief summary of our current knowledge of protective immunity to chlamydial urogenital infections of females. It is not meant to be exhaustive, but instead to touch upon aspects of protective immunity that have been described in both human and experimental animal models of chlamydial infection.

Gene knockout mice establish a primary protective role for major histocompatibility complex class II-restricted responses in Chlamydia trachomatis genital tract infection.

Mice with disrupted beta 2-microglobulin (beta 2m-/-), I-A (class II-/-), or CD4 (CD4-/-) genes were examined for their capacity to resolve Chlamydia trachomatis genital tract infection. C57BL/6 and beta 2m-/- mice resolved infection similarly and were culture negative by 4 to 5 weeks following infection. Conversely, major histocompatibility complex (MHC) class II-/- mice failed to resolve infection, and CD4-/- mice showed a significant delay (2 weeks). Secondary challenge of C57BL/6, beta 2m-/-, and CD4-/- mice established that acquired protective immunity, which was characterized by an infection of shortened duration and reduced shedding of infectious organisms, developed. Serological analysis of C57BL/6 and beta 2m-/- mice by enzyme-linked immunosorbent assays revealed no striking differences in the immunoglobulin subclass specificity of the anti-Chlamydia response, although some differences were observed in the magnitude of the immunoglobulin G2a (IgG2a) and IgG2b responses. Class II-/- mice produced lower-titered serum anti-Chlamydia antibodies of all isotypes. The serum antibody responses of CD4-/- mice were similar to those of C57BL/6 mice, except that the anti-Chlamydia IgA response was delayed by approximately 3 weeks. Analysis of vaginal washes for Chlamydia-reactive antibodies revealed the presence of IgG2a, IgG2b, and IgA in C57BL/6 and beta 2m-/- mice and primarily of IgA in CD4-/- mice. Vaginal washes from class II-/- mice were consistently antibody negative. Interestingly, the Chlamydia-specific IgA response in the vaginal washes of CD4-/- mice was delayed, but its appearance coincided with decreased shedding of infectious organisms and resolution of infection. Our results demonstrate that MHC class II-restricted T-cell responses are necessary for the development of protective immunity to Chlamydia genital tract infection and that local (vaginal) anti-Chlamydia IgA antibody coincides with the resolution of infection. A substantive role for MHC class I-restricted T-cell responses in protective immunity to Chlamydia genital tract infection was not confirmed.

Serologic responses of infertile women to the 60-kd chlamydial heat shock protein (hsp60).

OBJECTIVE: To determine whether women with Chlamydia trachomatis-associated tubal infertility are more likely than other infertile women to have antibodies to a particular region of the 60-kd chlamydial heat shock protein, hsp60. DESIGN: Serologic responses to the chlamydial hsp60 were examined in 43 infertile women seropositive for Chlamydia trachomatis, including 21 women with tubal infertility, 13 women with endometriosis, and 9 women with other causes of infertility. Antibody responses were localized to regions of hsp60 using five nonoverlapping recombinant polypeptides. RESULTS: Sixteen women with tubal infertility had anti-hsp60 antibodies compared with seven women with endometriosis and two women with other causes of infertility. Antibodies of 11 women with tubal infertility reacted predominantly with a region of hsp60 containing amino acids (201 to 300) compared with 1 women without tubal infertility. In contrast, antibodies that localized to the carboxyl terminus, amino acids (401 to 544), were seen equally in all groups. CONCLUSIONS: Among seropositive infertile women, antibodies that localized to amino acids (201 to 300) were immunodominant in those with tubal infertility but not in those with infertility due to other causes.

Identification of a 40-kDa human protein that cross-reacts with prokaryotic hsp60/chaperonins.

Cross-reactivity between the immunodominant bacterial hsp60 proteins and heterologous human target proteins has led to numerous hypotheses on the role of hsp60 in the pathogenesis of autoimmune disease. In this work, we describe a novel 40-kDa human protein that cross-reacts with bacterial hsp60 proteins. CCP40 (chaperone cross-reacting protein, 40-kDa) was identified in extracts from HL-60 (human promyelocytic leukemia) cells on Western blots probed with A57-E4, a monoclonal antibody specifying a linear polypeptide epitope common among the bacterial hsp60 proteins (Yuan et. al. (1992) Inf. Immun. 60, 2288-2296). CCP40 was detected in other human hematopoietic cell lines, but could not be detected in mature peripheral blood leukocytes. CCP40 was also expressed in human CD34+ peripheral blood progenitor cells, disappearing with cytokine-induced cellular maturation.

Reactivation of persistent Chlamydia trachomatis infection in cell culture.

Gamma interferon induces persistent chlamydial infections in cell culture. These infections are characterized by altered morphologic and biochemical features of the pathogen. These persistent forms are abnormally large and noninfectious and undergo unusual structural and functional changes, including production of a paucity of outer envelope constituents and normal levels of the chlamydial hsp60, an immunopathological antigen. The current investigation evaluates the events that occur during reactivation of infectious Chlamydia trachomatis from persistently infected cell cultures. Transfer of persistent chlamydial organisms to gamma interferon-free medium resulted in recovery of infectivity accompanied by an increase in levels of structural membrane proteins and reorganization of aberrant organisms to morphologically typical elementary bodies. In addition, reactivation of infectious organisms from persistent chlamydiae that were maintained in culture for several weeks was demonstrated. These studies show that persistent C. trachomatis maintains viability for extended periods, illustrate the reversibility of immunologically mediated persistent infections, and characterize reactivation at the ultrastructural and biochemical levels.