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The role of cytokines in chlamydial infection.

Cytokines are chemical substances produced by cells, which affect the function of other cells. Cytokines are important in generating inflammation and in sustaining the body’s immune response to infection. They may also be involved in the process of scarring and fibrosis that leads to the characteristic tissue damage associated with long term or severe chlamydial infection.

Chlamydial infection generates a cytokine response, both by direct infection of the epithelial cells lining the moist surfaces of the body (the mucosae), and by interaction with cells of the immune system [Fitzpatrick et al., 1991]. Thus, infection of epithelial cells derived from the human cervix with C. trachomatis, a major cause of genital tract infection, generates various cytokines (IL-1alpha , IL-8, GRO alpha, GM-CSF and IL-6) which help to generate and sustain an inflammatory response [Rasmussen et al., 1997]. The key event is probably chlamydial induced generation of the cytokine IL-1 alpha , which is able to stimulate the additional production of so-called pro-inflammatory cytokines by adjacent uninfected cells [Rasmussen et al., 1997]. Thus, the acute host response to Chlamydia at the mucosal surface may be initiated and sustained by epithelial cells, the prime targets of chlamydial infection [Rasmussen et al., 1997]. The proinflammatory cytokine IL-6, generated by either epithelial cells or by the interaction of chlamydiae with T lymphocytes of the cell mediated immune system, is probably important, together with IL-12, for sustaining the protective T-helper 1 cell mediated immune response [Williams et al., 1998]. A study of biopsy specimens taken from the eyes of 4 normal (uninfected) persons and from nine persons with trachoma showed that, in normal persons, there was little production of cytokines by the epithelial cells of the conjunctiva. In contrast, conjunctival epithelial cells from all of the subjects with trachoma showed a marked expression of the cytokines IL-1alpha and IL-1beta. Macrophages, one of the key effector cells of the host cell mediated immune response, were also observed deep in their ocular tissue, producing the inflammatory cytokine TNF alpha [Abu-el-Asrar et al., 1998]. In trachoma, blindness ultimately results from fibrosis, which gives rise to scarring and eventual distortion of the eyelids. It was noted that, in those individuals with extensive (severe) fibrosis, cells of the cell mediated immune system, (CD4⁺ T-helper lymphocytes), were present to a greater extent than antibody producing cells (B lymphocytes). It has been suggested that local production of cytokines as a direct or indirect result of C. trachomatis infection probably contribute to the conjunctival scarring and fibrosis characteristic of severe trachoma [Abu-el-Asrar et al., 1998].  

Desus-Babus et al., 2002 explored the interaction between infected epithelial cells and immune cells involved in the early stages of infection.  Incubation of monocyte-derived macrophages or of differentiated THP-1 cells with culture supernatants from C. trachomatis serovar E or L2-infected HeLa cells resulted in macrophage activation with a differential release of tumor necrosis factor alpha and upregulation of indoleamine 2,3-deoxygenase [a key enzyme in the inhibitory effect of interferon gamma on chlamydial growth in host cells]  but not of Toll-like receptor 2 and 4 mRNA expression [The Toll receptor is part of an evolutionary early immune system]. Co-cultivation of infected HeLa cells with differentiated THP-1 cells resulted in a reduction in chlamydial growth which was partially reversed by the addition of anti-TNF-alpha antibodies for serovar E or exogenous tryptophan for both serovars. Macrophage active oxygen radicals or the cytokines IL-8 or IL-1beta were apparently not involved in this inhibition. A gamma interferon-independent indoleamine 2,3-deoxygenase mRNA upregulation was also detected in dTHP-1 cells from infected cocultures. Supernatants from neutrophils added to the apical side of infected HeLa cell cultures were chlamydicidal, causing monocyte-derived macrophages to express antichlamydial activity. Neutrophils released more proinflammatory cytokines in response to serovar E rather than L2-infected cells. Nevertheless,  monocyte-derived macrophages were strongly activated by serovar L2 infected cells. The authors suggest that the early inflammatory response generated via cytokines with a nondisseminating or a disseminating strain may be different [Desus-Babus et al., 2002]. [Comment: Two serovars prove little. It will require further studies with a wider range of serovars to substantiate this hypothesis].

Cytokines and C. pneumoniae.

C. pneumoniae is associated with atherosclerotic plaque and is capable in vitro of transforming macrophages into foam cells, one of the characteristic cells of atheromatous plaque. Furthermore it is known that inflammation and the cumulative burden of infection is important in the progression of arterial disease [Bloemenkamp et al., 2002]. The question therefore is whether persistent infection of atherosclerotic plaque with C. pneumoniae and other infections generates a persisting pro-inflammatory cytokine response which exacerbates atherosclerosis and may eventually lead to coronary artery disease. This section focuses on some recent papers on this topic.

When freshly isolated peripheral blood mononuclear cells were stimulated for 24 hours with sonicated C. pneumoniae, significant amounts of the pro-inflammatory cytokines TNF-alpha and IL-1beta and of the anti-inflammatory cytokine IL-10 were released into the supernatant. Addition of serum increased C. pneumoniae-induced cytokine release two- to fivefold (p < 0.01), an effect which was not due to complement, mannose-binding lectin or lipopolysaccharide-binding protein. Neither the Toll-like receptor 4 (TLR4) lipopolysaccharide-binding CD14 played a significant role in this experimental system in the chlamydial induction of monocyte cytokines. However there was evidence that C. pneumoniae induced the serum-dependent pro-inflammatory cytokine TNF and IL-1beta response via Toll-like receptor 2. [Netea et al., 2002]. 

Dendritic cells are key antigen processing cells and play a central role in linking innate immunity (involving Toll-like receptors) and antigen-specific, cytokine stimulated, cell-mediated immune responses.  C. pneumoniae are taken up by dendritic cells but do not replicate to produce inclusions. However dendritic cells are potently stimulated by C. pneumoniae uptake as show by NF-kappaB activation. The cytokines IL-12p40 and TNF-alpha are secreted, and MHC class II molecules, CD40, CD80, and CD86 expression is up-regulated. This process is dependent on the presence of Toll-like receptor TLR2 and independent of TLR4 with the exception of IL-12p40 secretion [Prebeck et al., 2001].

Foam cells are essentially lipid-loaded macrophages and they are a major component of atherosclerotic lesions. Blessing et al., 2002 report that RAW 264.7 cells or mouse peritoneal macrophages loaded with oxidized or acetylated low density lipoproteins (LDL) to simulate foam cells, inhibit the growth of C pneumoniae but don't affect binding or uptake of the organism. Modified forms of LDL are not directly toxic to C pneumoniae and do not inhibit either the initial binding or internalisation of C pneumoniae by macrophages. Treatment of lipid-loaded macrophages with live, heat-killed, or UV-inactivated C pneumoniae stimulates secretion of cytokines and induced transcription of tumor necrosis factor-alpha in foam cells despite inhibition of nuclear factor-kappaB binding to DNA by prior treatment with oxidized LDL. It was conclude that chlamydial-induced foam cell formation is not conducive to the growth of C pneumoniae and does not inhibit the chlamydial-induced secretion of proinflammatory cytokines [Blessing et al., 2002].

The direct interaction of chlamydiae with endothelial cells lining vessel walls may promote atherogenesis by initiating endothelial procoagulant activity, increased monocyte adherence and increased cytokine production. It is known that both infection and atherogenesis is associated with the presence of inflammatory markers such as C-reactive protein and the cytokine IL-6. Visseren et al 2002 postulated that iron-mediated intracellular hydroxyl radical formation after infection might be a key event triggering endothelial production of interleukin-6 in vitro. Cultured endothelial cells were incubated with Fe⁺⁺ and Fe⁺⁺⁺ or infected with C. pneumoniae or influenza A/H1N1 virus for 48 and 24 hours, respectively. The involvement of iron or reactive oxygen species in IL-6 secretion was determined using the  H₂O₂ scavenger N-acetyl-l-cysteine, the intracellular hydroxyl radical scavenger dimethylthiourea or the iron chelator deferoxamine. Infection of endothelial cells with C. pneumoniae or influenza A induced an IL-6 response. Iron, the generation of intracellular hydroxyl radical and NF-kappaB activity were essential to this process, suggesting that reactive oxygen species generated in the Haber-Weiss reaction invoke an immunological response to infection of endothelial cells [Visseren et al., 2002]. Infection of vascular endothelial cells with C. pneumoniae also increases the expression of proatherogenic cytokines mediated by nuclear factor (NF)-kappaB, a transcription factor. Aspirin, salicylic acid, and 2 other unrelated NF-kappaB inhibitors showed a strong concentration-dependent inhibitory effect on chlamydial growth, indicated by the reduction of bacterial inclusions and the titer of infectious progeny. Aspirin also reduced the chlamydial induced secretion of IL-6 and IL-8. It was suggested that NF-kappaB-mediated gene activation represents a crucial step in the developmental cycle of C pneumoniae. Furthermore the cardioprotective activity of aspirin  might be due to its ability to inhibit C pneumoniae-induced NF-kappaB activation [Tiran et al., 2002].
 

C. pneumoniae also induces an IL-8 and TNF-alpha response in human lung carcinoma cells [Yang et al., 2003]. Inhibition of chlamydial DNA or protein synthesis did not affect this response, but heparin pretreatment of the chlamydiae did, suggesting that chlamydial adhesion to the host cell may be an important stimulus.

[MEW] February 2003

References

Abu-el-Asrar, A.M., Geboes, K., Tabbara, K.F., Al-Kharashi, S.A., Missotten, L., and Desmet, V. (1998). Immunopathogenesis of conjunctival scarring in trachoma. Eye 12, 453 - 460. [Good immunohistology study on conjunctival biopsies]

Blessing, E., Kuo, C. C., Lin, T. M., Campbell, L. A., Bea, F., Chesebro, B., Rosenfeld, M. E. (2002). Foam cell formation inhibits growth of Chlamydia pneumoniae but does not attenuate Chlamydia pneumoniae-induced secretion of proinflammatory cytokines. Circulation 105, 1976 - 1982.

Bloemenkamp, D. G., Mali, W. P., Tanis, B. C., Rosendaal, F. R. , van den Bosch, M. A., Kemmeren, J. M. et al.,  (2002). Chlamydia pneumoniae, Helicobacter pylori and cytomegalovirus infections and the risk of peripheral arterial disease in young women. Atherosclerosis 163, 149 - 156. [Interesting case control serology study].

Dessus-Babus, S., Darville, T. L., Cuozzo, F. P., Ferguson, K. & Wyrick, P. B. (2002). Differences in Innate Immune Responses (In Vitro) to HeLa Cells Infected with Nondisseminating Serovar E and Disseminating Serovar L2 of Chlamydia trachomatis. Infection and Immunity 70, 3234 - 3248.

Fitzpatrick, D. R., Wie, J., Webb, D., Bonfiglioli, R., Gardner, I. D., Mathews, J. D., and Bielefeldtohmann, H. (1991). Preferential binding of Chlamydia trachomatis to subsets of human-lymphocytes and induction of interleukin-6 and interferon- gamma. Immunology and Cell Biology  69, 337 - 348.

Gottsater, A., Forsblad, J., Matzsch, T., Persson, K., Ljungcrantz, I., Ohlsson, K. & Lindgarde, F. (2002). Interleukin-1 receptor antagonist is detectable in human carotid artery plaques and is related to triglyceride levels and Chlamydia pneumoniae IgA antibodies. Journal of  Internal Medicine 251, 61 - 68.

Netea, M. G., Kullberg, B. J., Galama, J. M., Stalenhoef, A. F., Dinarello, C. A., van der Meer, J. W. (2002). Non-LPS components of Chlamydia pneumoniae stimulate cytokine production through Toll-like receptor 2-dependent pathways. European Journal of Immunology 32, 1188 - 1195.

Prebeck, S., Kirschning, C., Durr, S., da Costa, C., Donath, B., Brand, K., Redecke, V., Wagner, H. & Miethke T. (2001). Predominant role of toll-like receptor 2 versus 4 in Chlamydia pneumoniae-induced activation of dendritic cells. Journal of Immunology 167, 3316 - 3323.  

Rasmussen, S. J., Eckmann, L., Quayle, A. J., Shen, L., Zhang, Y. X., Anderson, D. J., Fierer, J., Stephens, R. S. and Kagnoff, M. F. (1997). Secretion of proinflammatory cytokines by epithelial cells in response to Chlamydia infection suggests a central role for epithelial cells in chlamydial pathogenesis. Journal of Clinical Investigation  99, 77 - 87. Full article   

Tiran, A., Gruber, H. J., Graier, W. F., Wagner, A. H., van Leeuwen, E. B & Tiran, B. (2002). Aspirin inhibits Chlamydia pneumoniae-induced nuclear factor-kappa B activation, cytokine expression, and bacterial development in human endothelial cells. Arteriosclerosis Thrombosis and Vascular Biology 22, 1075 - 1080.

Visseren, F. L., Verkerk, M. S., van der Bruggen, T., Marx, J. J., van Asbeck, B. S. & Diepersloot, R. J. (2002). Iron chelation and hydroxyl radical scavenging reduce the inflammatory response of endothelial cells after infection with Chlamydia pneumoniae or influenza A. European Journal of Clinical Investigation 32, Suppl 1: 84 - 90. [Innovative study]

Williams, D. M., Grubbs, B. G., Darville, T., Kelly, K., and Rank, R. G. (1998). A role for interleukin-6 in host defense against murine Chlamydia trachomatis infection. Infection and Immunity 66, 4564 - 4567. Full article

Yang, J., Hooper, W. C., Phillips, D. J., Tondella, M. L., & Talkington, D. F. (2003). Induction of Proinflammatory Cytokines in Human Lung Epithelial Cells during Chlamydia pneumoniae Infection. Infection and Immunity 71, 614 - 620.

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