Heat Shock Protein 60 Specific T-Cell Response in Chlamydial Infections
Dr H.-M. Surcel, National Public Health Institute, Aapistie 1, Box 310, FIN-90101 Oulu, Finland. E-mail: email@example.com
Heat shock proteins (HSPs) of most pathogens, including Chlamydia, are major immune targets of both humoral- and cell-mediated immune mechanisms. During the last decade, many investigators have focused their research to elucidate the complex relationship of chlamydial HSPs, especially chlamydial HSP60, and the host immune response. A central issue is whether the pathologic mechanisms in chronic chlamydial diseases are associated with an enhanced immune response to chlamydial HSP60 which can mediate tissue destruction through cytotoxic reactions, or whether they are related to the Th2 type of response that eventually leads to partial or temporary suppression of an effective antichlamydial response. Our review highlights the available knowledge between immune responses to chlamydial HSP60 and chronic chlamydial infections in human.
Heat shock proteins (HSPs) are highly conserved proteins present practically in all prokaryotic and eukaryotic organisms. There are four main groups of structurally related HSPs based on their molecular weights (HSP90, HSP70 corresponding to DnaK of Escherichia coli, HSP60 to GroEL and small HSPs to GroES, respectively) and the individual members of each family, share 40–95% amino acid homology between different species [1–3]. They are important to both prokaryotic and eukaryotic cells in a variety of normal cellular functions, including their chaperone function during the intracellular (un)folding, assembly and translocation of newly synthetized or damaged proteins. Their role in cell survival and recovery from metabolic disturbances is reflected in the increased HSP synthesis induced by diverse forms of stress, such as infection, inflammation and exposure to harmful chemical or environmental factors [2, 4].
Despite their conservative nature HSPs are highly immunogenic. In addition to chlamydial infections, a number of infectious diseases are associated with activated humoral and cellular responses to microbial HSPs that has been extensively reviewed elsewhere [4, 5]. Owing to the high amino acid and structural homology of the HSPs between different species, the immune memory, either humoral- or cell-mediated, is considered to be limited not only to the microbe in question but also involve other, possibly more virulent pathogens that subsequently invade the host .
On the other hand, the immune response once initiated by the microbial HSP may also be evoked against autologous HSP epitopes. Recognition of the self-HSP may subsequently break down the immune tolerance against these cross-reactive structures and convert the protective immune responses into pathological ones . In this respect, the chlamydial HSPs, especially HSP60, have been a target of research interest during the past decade [6, 7].
Chlamydial infections and the expression of hsp60
Chlamydiae are obligate intracellular gram-negative-like bacteria that replicate in membrane-bound vacuoles (inclusions) in the cytoplasm of eukaryotic cells. The genus Chlamydia contains four species, C. trachomatis, C. pneumoniae, C. psittaci and C. pecorum, that differ in their host-cell tropism, but share certain biological properties in the course of their intracellular existence. Acute chlamydial infections in humans are often asymptomatic or mild oculogenital (C. trachomatis induced pelvic inflammatory disease (PID) or follicular conjunctivitis) or respiratory tract (C. pneumoniae) infections, which may resolve without adverse sequelae [9, 10]. These infections often recur or remain persistent, especially in the absence of treatment [11, 12]. The long-term consequences of recurrent or persistent chlamydial infections are severe leading to structural damage of the inflamed tissue. The best well-known examples thus far are C. trachomatis-related diseases, scarring of fallopian tubes in women with salpingitis and follicular conjunctiva of trachoma patients [13, 14]. Analogous influence on the affected organ by other chlamydial species has not been conclusively confirmed although the relationship of the C. pneumoniae infection with the development of atherosclerosis is intensively studied by several groups .
Persistent Chlamydia infection seems to involve incomplete chlamydial growth with a nonreplicative (or slowly replicating), noninfectious but metabolically active stage of development [14, 15]. In vitro, such nonproductive development has been demonstrated during stressful conditions, such as nutrient depletion, in the presence of penicillin or low levels of interferon (IFN)-γ. Therefore, it is not surprising that the production of chlamydial HSP60 in comparison to other chlamydial proteins increases during a persistent infection [16, 17].
Hsp60 antibodies and pathogenesis of chlamydial diseases
The enhanced expression of chlamydial HSP60 during persistent infection is indirectly seen as the appearance of HSP60-specific antibodies in the patient's serum (Table 1). The levels of chlamydial HSP60 antibodies are predictive for an increased risk of pelvic inflammatory disease in women . They also correlate with other pathologic sequelae of C. trachomatis infections, such as tubal factor infertility, habitual abortion, endometriosis, ectopic pregnancy [19–24], and scarring trachoma . Only a few reports have thus far been published of the antibody response to C. pneumoniae HSP60 [26–29].
Table 1. Examples of anti-chlamydial HSP60 (CHSP60) response in association with different chlamydial diseases in humans
|PID||Peeling et al. 1997 |
|TFI||Toye et al. 1993 |
|PID, occluded tubes||Eckert et al. 1997 |
|Infertility||Arno et al. 1995 |
|Ectopic pregnancy||Sziller et al. 1998 |
|TFI||Persson et al. 1999 |
|PID||Domeika et al. 1998 |
|Infertile couples||Witkin et al. 1998 |
|Infertile women||Freidank et al. 1995 |
|PID, ectopic pregnancy||Wagar et al. 1990 |
|TFI||Ault et al. 1998 |
|Chronic nongonococcal |
urethritis in men
|Horner et al. 1997 |
|Trachoma||Peeling et al. 1998 |
|Unstable angina pectoris||Bauriedel et al. 1999 |
|Atherosclerosis||Mayr et al. 1999 |
|Coronary artery disease||Jantos et al. 2000 |
|Asthma||Hahn et al. 2000 |
During persistent chlamydial infections the enhanced humoral response to chlamydial HSP60 is followed by a development of antibodies to human HSP60 [30, 31]. This has been interpreted as a sign of the autoimmune mechanisms underlying chlamydial disease exacerbation. However, conclusive evidence of the pathologic role or significance of HSP60 antibodies among other chlamydial antibodies is still lacking. We should not exclude the possibility that the enhanced humoral response to chlamydial HSP60 is simply a consequence of the intense stimulation of immune mechanisms by continuous or recurrent infections. This assumption is supported by the fact that HSP60 antibodies are often found concurrently with elevated antibody levels to the whole Chlamydia elementary body (EB) antigen [22, 23, 29, 31–33].
Hsp60 specific t-cell response in animals
Cell-mediated immunity (CMI) is essential in infection resolution and in protective immunity to Chlamydia but it is also thought to be involved in the immunopathogenesis of chlamydial diseases. This notion was originally based on animal studies, which showed that sensitization of guinea pigs with a Triton-X-100 soluble extract of chlamydial EBs accelerated an ocular inflammation characterized by accumulation of mononuclear lymphocytes . In a comparable study with monkeys, Taylor et al.  showed that the follicular response was observed only in C. trachomatis immunized but not in naive animals thereby giving an evidence for the significance of repeated chlamydial infection in terms of hypersensitivity response. Later Morrison and coauthors [36, 37] demonstrated that a 57-kDa protein, identified as chlamydial HSP60, is the active component and responsible for the inflammatory response in trachoma. Experimental studies in the monkey ‘pocket’ model of salpingitis have further supported the role of HSP60-specific T cells in the pathogenesis of salpingitis .
A possible generation of chlamydia induced autoimmune T-cell responses is the subject of debate. In a carefully performed study Yi et al.  demonstrated that in mice the proliferative activation of self-HSP60-responding T cells were seen only after concurrent immunization with mouse (i.e. self) and chlamydial HSP60. Immunization with the self-HSP60 alone induced T cells that secreted high level of anti-inflammatory interleukin (IL)-10 but showed antigen-specific anergy in terms of proliferative response. Importantly, during concurrent immunization with chlamydial HSP60, the cytokine secretion pattern of the self-HSP60 responding T cells switched to dominant production of pro-inflammatory IFN-γ showing that chlamydial infection can break tolerance of autoreactive cell reactions and lead them to participate in the inflammatory reactions during chlamydial disease.
Chlamydia hsp60 specific t-cell response in humans
Similar to the humoral responses, lymphocyte proliferative reactivity to chlamydial HSP60 is linked with repeated or chronic C. trachomatis infection (Table 2). Studying proliferative responses of circulating lymphocytes in vitro, Witkin et al.  demonstrated positive responses to chlamydial HSP60 more often in patients with salpingitis than in healthy controls and the response is directed to chlamydial peptides that share substantial homology with human HSP60 . More recently, Witkin and his study group reported that cell-mediated immunity to human HSP60 is associated with a history of spontaneous abortion .
Table 2. Examples of cellular responses to chlamydial (CHSP60) and human enter HSP60 in association with chronic chlamydial diseases in human
|CHSP60||Salpingitis||Witkin et al. 1993 |
|CHSP60 and |
|Salpingitis||Witkin et al. 1994 |
|Human HSP60||Spontaneous abortion, |
|Kligman et al. 1998 |
|CHSP60||Trachoma||Bailey et al. 1995 |
|CHSP60||Trachoma||Holland et al. 1993 |
|CHSP60||Trachoma||Holland et al. 1996 |
|CHSP60||Carotid plaque||Mosorin et al. 2000 |
|CHSP60||Atherosclerosis||Gaston et al. 2000 |
|CHSP60||Atherosclerosis||Curry et al. 2000 |
While an enhanced cell-mediated response to HSP60 is unfavourable in women with pelvic inflammatory disease, (PID) related diseases [40–42], the situation seems opposite in trachoma patients. According to Bailey and coauthors , lymphocyte proliferation to chlamydial antigens, including major outer membrane protein (MOMP) and HSP60 is enhanced in individuals who spontaneously resolve ocular chlamydial infection compared to those with persistent ocular disease. Holland et al.  have further shown that scarring trachoma is linked with markedly depressed lymphocyte proliferation response to chlamydial antigens and involves the downregulation of Th1 activity and the IFN-γ response . Interestingly, activation of chlamydial HSP60-specific Th2 cells was seen in individuals with severe scarring trachoma, but not in age- and sex-matched controls. This supports a hypothesis that chlamydial pathogenesis involves activated Th2 cells and suggests that chlamydial HSP60 plays a special role in this respect.
In order to better understand the significance of HSP60-responding T cells in inflamed tissue, we have derived Chlamydia-specific T-cell lines from the obstructed fallopian tubes of infertile women (TFI) and from endometrial biopsies of acute PID patients. According to our results, Chlamydia-reactive T cells were present in both PID and TFI patients, while chlamydial HSP60-reactive T cells were only found in salpingeal tissue . This was the first direct evidence in humans to show that chlamydial HSP60-reactive T cells can be found among the in vivo activated T cells and that they participate in the inflammatory process in salpingeal tissue.
In order to obtain further information about the frequency or relative importance of chlamydial HSP60 as a T-cell-stimulating antigen in the tissue, we used a limiting dilution method to derive T-cell clones from C. trachomatis-specific T-cell cultures. Although chlamydial elementary body (EB) contains dozens of proteins that are able to elicit lymphocyte proliferation , as many as 30% of the C. trachomatis-specific T-cell clones recognized chlamydial HSP60, suggesting that it is a relatively important antigen for the induction of cellular responses in the salpingeal tissue of TFI patients [Kinnunen and coauthors, unpublished observations].
In analogous experiments with C. pneumoniae reactive T-cell lines derived from atherosclerotic plaques of the carotid artery we  and others [49, 50] found that chlamydial HSP60 induces proliferative response in some C. pneumoniae reactive T-cell lines showing that chlamydial HSP60 is participating in the activation of cell-mediated mechanisms in the atherosclerotic plaques. On the other hand, we observed that antigenic peptides on human HSP60 are only recognized in one out of five cases of chlamydial HSP60 specific T-cell lines derived from different individuals . This suggests that autoimmune responses, although possible, would not play a dominant role in the Chlamydia HSP60-induced T-cell activation. The result is in line with corresponding data on T-cell responses in Yersinia-induced human arthritis, where one out of four Yersinia HSP60-reactive T cells recognized a specific epitope in a human counterpart as well .
Chlamydial hsp60 and the cytokine response
IFN-γ production has been identified as one of the main factors in protective immunity, and in vitro experiments have shown that IFN-γ is also important in the development of chronic chlamydial infection [6, 14]. According to the present view, a lack of IFN-γ, i.e. downregulation of the Th1 response, at the site of inflammation during a chlamydial infection could lead to prolonged infection and inflammation . Immunosuppression of Th1 activity is probably a multifactorial event, but in terms of HSP60 responses, IL-10, an antagonistic cytokine of IFN-γ[53, 54], is interesting. An enhanced IL-10 secretion, i.e. a shift towards the Th2 type of cellular response has been shown in mice to associate with lymphocyte activation against self-Hp60-antigen  and to lead to a delayed resolution of the chlamydial infection .
Whether a comparable relationship of Chlamydia-induced Th2 activity and self-HSP60 reactivity is playing a immunoregulative role in humans is not known. It is tempting to speculate that the decreased cell-mediated immunity, which Holland et al.  have already linked with chlamydial HSP60 reactivity in trachoma, could be a consequence of self-HSP60-responding T-cell activation. According to our results with salpingeal lymphocytes, about half of the chlamydial HSP60 responding T cells produced IL-10, either alone or in concert with IFN-γ[Kinnunen and coauthors, unpublished observations]. We think that the observed frequency of IL-10 secreting T cells is relatively high and possibly reflects their capacity to reduce the Th1 response and the effect of IFN-γ that is needed to eradicate C. trachomatis infection in the salpingeal tissue.
A key issue in chlamydial diseases is whether the pathologic mechanisms are associated with an enhanced immune response mediating tissue destruction through cytotoxic reactions, or whether they are related to the Th2 type of response that eventually leads to the partial or temporary suppression of an effective antichlamydial response. In both models, chlamydial HSP60 continues to be the key antigen. In general, earlier reports describing an enhanced immune responses to Chlamydia, and especially chlamydial HSP60, during chronic chlamydial diseases (reviewed in ), consider the responses to be related to the antigenic mimicry of HSP60 proteins, which triggers an autoimmune response against self-HSP60 and, as such, is an aberration of the host-immune system. During recent years, however, several experimental animal studies have been published focusing on the possibility that the self-HSP60 reactivity observed after chlamydial infection may induce Th2 responses and, consequently, delay the resolution of the infection [52, 55]. In the latter model of Chlamydia immunity, the self-HSP60 reactivity reflects a regulative process of the host to protect itself from over-reacting to inflammatory stimuli. It is also possible that both are right and that the stage of equilibrium is determined by as yet undefined factors such as the type of infected cells or the HLA genotype of the host.
More studies are needed to elucidate the characteristics of HSP60-induced immune response in chlamydial diseases and to investigate its role or significance in the development of an adverse outcome of the infection. We consider especially interesting the results of circulating human HSP60 antibodies and antigen having been demonstrated in the peripheral circulation of normal individuals in varying concentrations, which have generally been higher in women than in men . On the other hand, in an animal model, immunization with self-HSP60 can be used therapeutically to reduce immune response to foreign antigens . There are no studies where pre-existing self-HSP60 antigen in circulation would have been analyzed in patients with acute Chlamydia infection but it is tempting to speculate that elevated level of self-HSP60 antigen would interfere in the immune response to Chlamydia and thereby influence in the outcome of chronic chlamydial infection.
We thank Dr Matti Lehtinen for critically reading the manuscript.