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The Journal of Immunology, 2008, 180, 1158 -1168
Copyright © 2008 by The American Association of Immunologists, Inc.
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The Proinflammatory Cytokine Response to Chlamydia trachomatis Elementary Bodies in Human Macrophages Is Partly Mediated by a Lipoprotein, the Macrophage Infectivity Potentiator, through TLR2/TLR1/TLR6 and CD141

Sylvette Bas2,*, Laurence Neff*,{dagger}, Madeleine Vuillet*, Ursula Spenato*, Tsukasa Seya{ddagger}, Misako Matsumoto{ddagger} and Cem Gabay*,{dagger}

* Division of Rheumatology, Department of Internal Medicine, University Hospital, and {dagger} Department of Pathology and Immunology, Geneva Medical School, Geneva, Switzerland; and {ddagger} Department of Microbiology and Immunology, Hokkaido University Graduate School of Medicine, Sapporo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Chlamydiae components and signaling pathway(s) responsible for the production of proinflammatory cytokines by human monocytes/macrophages are not clearly identified. To this aim, Chlamydia trachomatis-inactivated elementary bodies (EB) as well as the following seven individual Ags were tested for their ability to induce the production of proinflammatory cytokines by human monocytes/macrophages and THP-1 cells: purified LPS, recombinant heat shock protein (rhsp)70, rhsp60, rhsp10, recombinant polypeptide encoded by open reading frame 3 of the plasmid (rpgp3), recombinant macrophage infectivity potentiator (rMip), and recombinant outer membrane protein 2 (rOmp2). Aside from EB, rMip displayed the highest ability to induce release of IL-1β, TNF-{alpha}, IL-6, and IL-8. rMip proinflammatory activity could not be attributed to Escherichia coli LPS contamination as determined by the Limulus Amoebocyte lysate assay, insensitivity to polymyxin B (50 µg/ml), and different serum requirement. We have recently demonstrated that Mip is a "classical" bacterial lipoprotein, exposed at the surface of EB. The proinflammatory activity of EB was significantly attenuated in the presence of polyclonal Ab to rMip. Native Mip was able to induce TNF-{alpha} and IL-8 secretion, whereas a nonlipidated C20A rMip variant was not. Proinflammatory activity of rMip was unaffected by heat or proteinase K treatments but was greatly reduced by treatment with lipases, supporting a role of lipid modification in this process. Stimulating pathways appeared to involve TLR2/TLR1/TLR6 with the help of CD14 but not TLR4. These data support a role of Mip lipoprotein in pathogenesis of C. trachomatis-induced inflammatory responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Chlamydiae are important human pathogens due to the wide repertoire of important diseases that they cause (1, 2, 3, 4, 5). These microorganisms infect primary epithelial cells with subsequent attraction of monocytes/macrophages (6, 7). They frequently cause chronic inflammatory diseases characterized by the presence of a high number of mononuclear cells (8) that are involved in the pathogenesis by inducing mediators of inflammation. However, few data have been published about chlamydiae components involved in inflammatory response, and results are still debated. Chlamydial LPS (9, 10) and heat shock protein (hsp)360 (11, 12) were involved in some reports but not in others (13, 14, 15). In signaling pathway, whole bacteria or Chlamydia hsp60 have been shown to induce TLR-mediated activation, but the signaling receptor differed among studies from both TLR2 and TLR4 (16), only TLR4 (17, 18), only TLR2 (14), to largely TLR2 and to a minor extent TLR4 (19). Except LPS and hsp60, chlamydiae components responsible for these effects were unidentified.

To identify chlamydial component(s) able to induce production of proinflammatory cytokines by human monocytes/macrophages, inactivated elementary bodies (EB), one of the two forms presented by chlamydiae in their biphasic developmental cycle, as well as seven individual chlamydial Ags: purified LPS, recombinant hsp70 (rhsp70), rhsp60, rhsp10, recombinant polypeptide encoded by open reading frame 3 of the plasmid (rpgp3), recombinant macrophage infectivity potentiator (rMip), and recombinant outer membrane protein 2 (rOmp2) were carefully purified and tested. Aside from EB, rMip displayed the highest proinflammatory activity, stimulating the synthesis of IL-1β, TNF-{alpha}, IL-6, and IL-8. Mip was recently shown to have lipid modification similar to that of other procaryotic lipoproteins and to be exposed at the surface of EB (20). The proinflammatory activity of EB was significantly attenuated in the presence of polyclonal Ab to rMip. Failure of stimulation with a nonlipidated C20A rMip variant as well as after lipase treatment of rMip showed that the proinflammatory activity was dependent upon lipid modification. Use of blocking mAb and human embryonic kidney (HEK)-293 transfected cells revealed that TLR2/TLR1/TLR6 and CD14 but not TLR4 were involved in mediating these effects.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Bacteria and Ags

EB of Chlamydia trachomatis LGV2 strain 434 (inactivated by a photochemical treatment affecting bacterial genomes) were either purchased from Biodesign International (Milan Analytica) or prepared according to the method of Boleti et al. (21). Chlamydia LPS was extracted according to Rund et al. (22). LPS from Escherichia coli serotype O55:B5 and Salmonella typhimurium were purchased from Sigma-Aldrich (Fluka Chemie) and repurified (23) to rule out protein and lipoprotein contamination. Cloning, expression in E. coli, and purification of six different recombinant proteins from C. trachomatis (rhsp70, rhsp60, rhsp10, rpgp3, rMip, and rOmp2) were previously described (24, 25). To rule out LPS and LPS-associated molecule contamination (26, 27, 28), all recombinant proteins were subsequently treated by polymyxin B-agarose (Sigma-Aldrich) and had <10 endotoxin units per milligram, according to the Limulus Amoebocyte Lysate chromogenic assay (BioWhittaker Cambrex), which is an amount that did not stimulate proinflammatory cytokine production by itself. Preparations of native Mip and C20A rMip variant were previously described (20). Protein concentrations were determined by using a microbicinchoninic acid protein assay kit (Pierce, Perbio Science). Racemic Pam3CSK4 (Pam3-Cys-Ser-Lys4-OH) and Pam2CSK4 (Pam2-Cys-Ser-Lys4-OH) used as synthetic triacylated and diacylated control lipopeptides, respectively, were obtained from EMC Microcollections.

Abs and antiserum preparation

Anti-CD14 (clone MY4, IgG2b) was purchased from Coulter Clone (Beckman Coulter), anti-TLR1 (clone GD2.F4, IgG1) from Hycult Biotechnology (Biocoba), anti-TLR2 and anti-TLR4 (clone TL2.1 and HTA125, respectively, IgG2a) from ImmunoKontact (AMS Biotechnology). Anti-TLR6 (clone 127, IgG1) was previously described (29). Isotype-matched mouse IgG1 control was purchased from Southern Biotechnology Associates and IgG2a, and IgG2b from Coulter Clone. Preparation of IgG fraction from rMip antiserum was previously described (20).

Human monocyte/macrophage culture

PBMC from healthy blood donors were isolated by density gradient centrifugation with Ficoll-Hypaque (Amersham Biosciences). Monocytes/macrophages were separated by aggregation, gradient of FBS (Invitrogen Life Technologies), and rosetting (30, 31). Monocyte purity consisted of ≥90% CD14+ cells, ≤1% CD3+ cells, and ≤1% CD19+ cells as assessed by flow cytometry. The enriched monocytes were cultured in 24-well plates (105 cells/1 ml per well) in RPMI 1640 containing 2 mM GlutaMAX I, supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, and heat-inactivated (30 min at 56°C) endotoxin-free 10% (v/v) FBS (Invitrogen Life Technologies). After 48 h (or indicated time periods) stimulation at 37°C with indicated stimuli, cultures were centrifuged at 400 x g for 10 min at 4°C and cell-free supernatants were collected and stored at –70°C until cytokine measurements.

THP-1 cell culture

Cells of the human promyelomonocytic cell line THP-1 (32) were purchased from American Type Culture Collection and were grown in RPMI 1640 medium as described above. For monocytic differentiation, they were seeded in 24-well flat-bottom tissue culture plates at a density of 2.5 x 105 cells/1 ml per well and allowed to adhere and differentiate 48 h at 37°C in presence of 10 nM PMA (Sigma-Aldrich). After repeated washing with RPMI 1640, PMA-differentiated THP-1 cells were stimulated at 37°C with indicated stimuli. Cell-free supernatants were harvested after 4 h (or indicated time periods) of incubation and kept at –70°C until cytokine measurements.

Native Mip blocking experiments

PMA-differentiated THP-1 cells were pretreated at 37°C for 1 h with 50 µg/ml human IgG to block Fc receptors and prevent subsequent nonspecific binding of IgG and then stimulated with inactivated C. trachomatis EB (5 x 106/ml) in presence of rabbit polyclonal anti-rMip IgG or preimmune IgG (0, 40, or 80 µg/ml). Cell-free supernatants were harvested after 4 h of incubation and kept at –70°C until cytokine measurements.

Heat, proteinase K, alkaline, and lipase treatments of rMip

Heat sensitivity was determined by heating rMip at 100°C for 3 h and proteinase K sensitivity by incubating rMip with 100 µg/ml proteinase K (Promega) in 100 mM Tris-HCl (pH 8.0) at 37°C for 2 h, followed by addition of 200 µM PMSF. Alkaline hydrolysis was performed according to Muhlradt and Frisch (33). Briefly, rMip solution was adjusted to pH 13.0 with sodium hydroxide, incubated at 37°C for 1 h, and neutralized with HEPES before assay. Lipase sensitivity was determined with two glycerol ester hydrolases (E.C. 3.1.1.3.) having different side chain specificity (34). Either 1000 U/ml pig pancreas lipase (type VI-S lipase; Sigma-Aldrich) or 10,000 U/ml Rhizopus arrhizus lipase (type XI lipase; Sigma-Aldrich) were added to rMip at 37°C for 16 h, in 50 mM HEPES buffer (pH 7.5), 10 mM CaCl2, followed by heating at 100°C before assay (35). As controls, rMip was incubated in buffer with no sodium hydroxide or enzymes and PMA-differentiated THP-1 cells were incubated with buffer and enzymes in the absence of rMip. Cell-free supernatants were harvested after 4 h of incubation and kept at –70°C until cytokine measurements.

CD14, TLR1, TLR2, TLR4, and TLR6 blocking experiments

Blocking experiments were performed after 1 h pretreatment of PMA-differentiated THP-1 cells with 50 µg/ml human IgG to block Fc receptors and prevent subsequent nonspecific binding of blocking Ab or nonimmune isotype controls. Cells were next incubated at 37°C for 1 h with blocking Ab (5 µg/ml) before stimulation with either 1 µg/ml rMip, inactivated C. trachomatis EB (5 x 106/ml), 0.01 µg/ml lipopeptides (Pam3CSK4 or Pam2CSK4), or 1 µg/ml E. coli LPS. Cell-free supernatants were harvested after 4 h of incubation and kept at –70°C until cytokine measurements.

Response of TLR/CD14 cell lines

HEK-293 cells stably transfected with either the empty plasmid (293-Null) or human TLR1/2, TLR2/6, or TLR2/CD14 genes were purchased from InvivoGen (LabForce) and maintained in DMEM (Invitrogen Life Technologies) supplemented with 4.5 g/L glucose, 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 10 µg/ml blasticidin S (InvivoGen) for 293-Null, 293-hTLR1/2, and 293-hTLR2/6 and with 50 µg/ml HygroGold (InvivoGen) for 293-hTLR2/CD14. For stimulation experiments, stable transfected cells were seeded into individual wells of a 48-well tissue culture plate at a concentration of 3 x 105 cells in 300 µl of complete medium and allowed to adhere overnight. The following day, fresh medium was added, and the cells were stimulated with either 1 µg/ml rMip, inactivated C. trachomatis EB (5 x 106/ml), 1 or 0.01 µg/ml lipopeptides (Pam3CSK4 or Pam2CSK4), or 1 µg/ml E. coli LPS for 24 h. Culture supernatants were collected, and IL-8 content was analyzed. Results were expressed in terms of fold increase over the IL-8 levels of unstimulated cells.

Cytokine measurements

Extracellular release of IL-1β, TNF-{alpha}, IL-6, and IL-8 was determined by a sandwich ELISA technique using the DuoSet ELISA Development Systems (R&D Systems), according to the manufacturer’s instructions. The ELISA detection limits were 2 pg/ml for all tested cytokines. When the distributions in cytokine production were not normal, results were expressed as median and interquartile range.

Statistical analysis

Statistical analysis was performed using the SPSS statistical software (for Macintosh, v.10). Kruskal-Wallis and Mann-Whitney U tests were used to compare the levels of inflammatory cytokines produced by human monocytes/macrophages in response to various microbial components. A comparison between two groups was made only when the Kruskal-Wallis test yields statistically significant results. Statistical analysis for PMA-differentiated THP-1 cell and HEK assays were performed using a Student’s t test. Differences were considered significant at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Inactivated C. trachomatis EB and rMip elicited proinflammatory cytokine production by human monocytes/macrophages and PMA-differentiated THP-1 cells

The pathology of C. trachomatis infection seems to be related to chronic inflammation, characterized by the dominating presence of macrophages in injured tissues. We have therefore tested the ability of inactivated EB and seven chlamydial components (LPS, rhsp70, rhsp60, rhsp10, rpgp3, rMip, and rOmp2) to stimulate the production of various proinflammatory cytokines by healthy blood donor monocytes/macrophages. Inactivated EB as well as rMip induced the release of IL-1β, TNF-{alpha}, IL-6, and IL-8 in contrast to other chlamydial Ags that did not display any consistent stimulatory effects. Both E. coli and S. typhimurium LPS, used as positive controls, highly induced the release of proinflammatory cytokines, in contrast to chlamydial LPS, as previously reported (10, 15, 36, 37) (Fig. 1). To further define the ability of rMip to stimulate the synthesis of proinflammatory cytokines, dose-response and time course experiments were performed. A dose-dependent stimulation was observed in presence of increasing concentrations of rMip (0.005–5 µg/ml) (Fig. 2). The release of TNF-{alpha} was time-dependent with maximal levels being reached after 8 h of culture (Fig. 3). Our data agree with previous findings showing that monocytes/macrophages produced TNF-{alpha} shortly after stimulation with lipoproteins (38, 39).


Figure 1
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  		FIGURE 1. Cytokine productions by human monocytes/macrophages in response to various bacterial Ags. Human monocytes macrophages (105 cells/1 ml per well) were stimulated by inactivated C. trachomatis EB (5 x 106/ml), rhsp70, rhsp60, rhsp10, rpgp3, rOmp2, rMip, LPS, E. coli LPS or S. typhimurium LPS at 1 µg/ml. After 48 h of culture, supernatants were collected and their content in IL-1β, IL-6, IL-8, and TNF-{alpha} were analyzed by ELISA. Results were obtained from three different cultures performed in triplicates. Horizontal bar within boxes shows the median, boxes show the interquartile range, and vertical bar shows the 95% confidence interval (values above and below these levels were plotted separately). *, p < 0.05; **, p < 0.005 determined by comparison with unstimulated cells using Mann-Whitney U test.

 

Figure 2
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  		FIGURE 2. Cytokine productions by human monocytes/macrophages in response to increasing concentrations of rMip. Each value represents the mean ± SD of triplicates from two experiments. *, p < 0.05; **, p < 0.005; ***, p < 0.0001 determined by comparison with unstimulated cells using Mann-Whitney U test.

 

Figure 3
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  		FIGURE 3. Kinetics of TNF-{alpha} production by human monocytes/macrophages in response to rMip (1 µg/ml). Each value represents the mean ± SD of triplicates from three experiments. *, p < 0.05; **, p < 0.005; ***, p < 0.0001 determined by comparison with unstimulated, cells at the same time point, using Mann-Whitney U test.

 
As PMA-differentiated THP-1 cells are resembling to tissue macrophages and are commonly used as models for human monocytes/macrophages (40), they were also tested for their ability to produce cytokines in presence of the bacterial components. As already observed with human monocytes/macrophages, inactivated EB as well as rMip were the most effective stimuli in triggering the synthesis of IL-1β, TNF-{alpha}, IL-6, and IL-8. Hsp60 triggered a lower but significant synthesis of IL-1β, TNF-{alpha}, IL-6, and IL-8. Hsp70 triggered a very low but significant synthesis of IL-6, and pgp3 and Chlamydia LPS stimulated a low IL-8 production, whereas E. coli and S. typhimurium LPS strongly stimulated the synthesis of all the cytokines (Fig. 4). Because rMip was the only chlamydial Ag that consistently stimulated THP-1 cells, dose-response and time course experiments were performed with rMip. At concentrations ranging from 0.001 to 10 µg/ml, rMip elicited a dose-dependent increase in IL-1β, TNF-{alpha}, and IL-8 production with maximal response at 10 µg/ml. The production of cytokines was significantly stimulated when cells were incubated with 1 µg/ml rMip (Fig. 5). When comparing Figs. 2 and 5, the proinflammatory activity of rMip was weaker in PMA-differentiated THP-1 cells than in primary human monocytes/macrophages. This difference might be due to different levels of TLRs or CD14 expression at the surface of the cells. In PMA-differentiated THP-1 cells, significant stimulation of TNF-{alpha} release was already detected within 2 h and increased rapidly thereafter. At 24 h, TNF-{alpha} levels reached peak values and then declined gradually (Fig. 6).


Figure 4
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  		FIGURE 4. Cytokine productions by PMA-differentiated THP-1 cells in response to various bacterial Ags. THP-1 cells (2.5 x 105 cells/1 ml per well) were cultured with 10 nM PMA for 48 h and then stimulated by inactivated C. trachomatis EB (5 x 106/ml), rhsp70, rhsp60, rhsp10, rpgp3, rOmp2, rMip, LPS, E. coli LPS, or S. typhimurium LPS at 1 µg/ml. After 48 h of culture, supernatants were collected and their content in IL-1β, IL-6, IL-8, and TNF-{alpha} were analyzed by ELISA. Results were obtained from two different cultures performed in triplicates. Each value represents the mean ± SEM. *, p < 0.05; **, p < 0.005; ***, p < 0.0001 determined by comparison with unstimulated cells using Student’s t test.

 

Figure 5
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  		FIGURE 5. Cytokine productions by PMA-differentiated THP-1 cells after 4 h stimulation with increasing concentrations of rMip. Each value represents the mean ± SD of triplicates from three experiments. **, p < 0.005; ***, p < 0.0001 determined by comparison with unstimulated cells using Student’s t test.

 

Figure 6
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  		FIGURE 6. Kinetics of TNF-{alpha} production by PMA-differentiated THP-1 cells in response to rMip (1 µg/ml). Each value represents the mean ± SD of triplicates from one representative experiment. **, p < 0.005; ***, p < 0.0001 determined by comparison with unstimulated cells, at the same time point, using Student’s t test.

 
Because the response of human monocytes/macrophages varied from one donor to another, the proinflammatory activity of rMip was further studied on PMA-differentiated THP-1 cells to obtain more homogeneous and reproducible data. Based on above results and for practical reasons, the effect of rMip was further examined at 1 µg/ml concentration and at the shortest time point (4 h) required to obtain a significant production of TNF-{alpha}.

E. coli LPS contamination is not involved in the production of TNF-{alpha} mediated by rMip

Despite the fact that no endotoxin was detected by the Limulus Amoebocyte lysate assay in the highly purified preparation of rMip, the possibility that its proinflammatory activity could be attributed to E. coli LPS contamination was further investigated by testing rMip sensitivity to polymyxin B (50 µg/ml). The ability of rMip to induce TNF-{alpha} production by PMA-differentiated THP-1 cells was unaffected by the presence of polymyxin B whereas highly purified E. coli LPS was unable to induce TNF-{alpha} production in presence of polymyxin B. In addition, rMip and LPS differed in their serum requirement: in absence of serum, TNF-{alpha} production induced by rMip was significantly increased whereas E. coli LPS was devoid of effect as already reported (41, 42, 43, 44) (Fig. 7).


Figure 7
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  		FIGURE 7. Effect of polymyxin B and FBS on the ability of rMip and E. coli LPS to induce TNF-{alpha} production by PMA-differentiated THP-1 cells. Cells (2.5 x 105 cells/1 ml per well) were cultured with 10 nM PMA for 48 h and then stimulated by rMip or E. coli LPS (1 µg/ml) in the presence or absence of 50 µg/ml polymyxin B sulfate, and in the presence or absence of 10% FBS. After 4 h stimulation, supernatants were collected and their content in TNF-{alpha} were analyzed by ELISA. Each value represents the mean ± SD of triplicates from two experiments for polymyxin assay and from four experiments for FBS assay. *, p < 0.05; ***, p < 0.0001 determined by comparison with medium alone using Student’s t test.

 
Anti-rMip polyclonal IgG partly inhibit the production of TNF-{alpha} mediated by C. trachomatis EB

The probable involvement of native Mip in initiation of chlamydial infections has been demonstrated by Lundemose et al. (45) who observed a neutralization of the organism in cell culture in presence of anti-Mip Ab. To investigate whether the proinflammatory activity of C. trachomatis EB could be attributed to the presence of native Mip exposed at the EB surface (20), experiments were conducted in presence and in absence of rabbit polyclonal IgG anti-rMip. The presence of 80 µg/ml IgG anti-rMip led to significant inhibition of TNF-{alpha} release (33%, p = 0.014) when compared with TNF-{alpha} release in presence of preimmune rabbit IgG (Fig. 8). These results show that native Mip exposed at the EB surface contributes to induce TNF-{alpha} production when PMA-differentiated THP-1 cells are cultured in presence of C. trachomatis EB.


Figure 8
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  		FIGURE 8. Effect of increasing concentrations of anti-rMip polyclonal IgG upon the production of TNF-{alpha} mediated by C. trachomatis EB. THP-1 cells (2.5 x 105 cells/1 ml per well) were cultured with 10 nM PMA for 48 h, preincubated for 1 h with 50 µg/ml human IgG to block Fc receptors to prevent nonspecific binding of blocking Ab or preimmune IgG, and then stimulated by inactivated C. trachomatis EB (5 x 106/ml). Stimulation was performed in presence of 0, 40, or 80 µg/ml of either rabbit polyclonal IgG anti-rMip or preimmune rabbit IgG. After 4 h of stimulation, supernatants were collected and their content in TNF-{alpha} were analyzed by ELISA. Results were obtained from two different cultures performed in triplicates. Each value represents mean ± SEM. *, p < 0.05 determined by comparison with EB activation in presence of preimmune rabbit IgG.

 
Native Mip but not C20A rMip variant elicited proinflammatory cytokine production

To ascertain that wild-type (WT) rMip activity featured the same stimulatory properties than native Mip, this lipoprotein was purified from C. trachomatis EB by immunoprecipitation and tested for its ability to induce the production of TNF-{alpha} by PMA-differentiated THP-1 cells. In addition, to assess the importance of lipid modification, proinflammatory activity of nonlipidated C20A rMip variant was also tested. As shown in Fig. 9, native Mip was able to induce release of TNF-{alpha} and IL-8, in a similar amount than WT rMip. In contrast, no cytokine production was observed when THP-1 cells were cultured in presence of C20A rMip variant suggesting that lipidation plays a major role in proinflammatory activity of native and WT rMip.


Figure 9
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  		FIGURE 9. Cytokine productions by PMA-differentiated THP-1 cells in response to various Mip preparations. THP-1 cells (2.5 x 105 cells/1 ml per well) were cultured with 10 nM PMA for 48 h and then stimulated by native Mip, rMip, or nonlipidated C20A rMip variant at 1 µg/ml. After 4 h of stimulation, supernatants were collected and their content in IL-8 and TNF-{alpha} were analyzed by ELISA. Results were obtained from two different cultures performed in triplicates. Each value represents mean ± SEM. **, p < 0.005; ***, p < 0.0001 determined by comparison with native Mip stimulated cells.

 
Production of proinflammatory cytokines induced by rMip is lipid dependent

Because rMip appeared to have the same proinflammatory activity that native Mip and because it is not feasible to purify adequate amount of native Mip from C. trachomatis for analysis, WT rMip, previously shown to be lipidated as native Mip (20), was used for further investigation. To determine the biochemical nature of rMip proinflammatory activity, experiments were conducted to examine the possible involvement of protein and lipid parts of rMip. To determine the importance of the total protein part, attempts to destroy rMip activity by heat, and proteinase K were tested. Heat treatment was unsuccessful, indicating that protein’s native conformation is not essential for rMip stimulatory activity. Digestion with proteinase K resulted in loss of the 27.6- and 32-kDa bands (20) (data not shown) but did not affect proinflammatory activity. This result suggests that rMip activity may still reside in resulting lipopeptides after proteolytic digestion, as was already reported for a macrophage-activating lipopeptide from Mycoplasma fermentans (46). The involvement of the lipopeptide moiety was confirmed by three different treatments releasing ester-linked fatty acids. Alkaline hydrolysis completely abolished rMip activity and treatment with lipases from two different sources led to significant losses of TNF-{alpha} release (76% with pig pancreas and 78% with R. arrhizus lipases, p < 0.05) (Fig. 10). No significant difference was observed between untreated and mock-treated PMA-differentiated THP-1 cells (data not shown). The substitution of rMip with fatty acids in an ester linkage, alkali-labile (47), appears therefore to be crucial for its proinflammatory activity, as reported for other lipoproteins or lipopeptides (33, 48, 49, 50, 51). These data confirm that lipid modification of rMip is essential for its ability to stimulate production of proinflammatory cytokines by PMA-differentiated THP-1 cells.


Figure 10
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  		FIGURE 10. Effect of heat, proteinase K, alkaline hydrolysis, and lipase treatments of rMip on TNF-{alpha} production by PMA-differentiated THP-1 cells. Cells were stimulated by untreated or treated rMip (1 µg/ml). rMip was either heated at 100°C during 3 h, or incubated at 37°C for 2 h with proteinase K (100 µg/ml), or treated for 1 h with sodium hydroxide (pH 13) before HEPES neutralization (33 ), or incubated at 37°C for 16 h with pig pancreas or Rhizopus arrhizus lipases. Digests were either treated by 200 µM PMSF or heated at 100°C before assay. Other conditions were described above. Each value represents the mean ± SD of triplicates from two experiments. *, p < 0.05; ***, p < 0.0001 determined by comparison with untreated rMip using Student’s t test.

 
CD14, TLR1, TLR2, and TLR6 but not TLR4 are involved in rMip activation of THP-1 cells

If rMip is able to induce proinflammatory cytokines in human monocytes/macrophages, the specific host receptors that mediate its activation is unknown. As TLRs and CD14 are usually involved in bacterial lipoprotein recognition (52), experiments were conducted in the presence and in the absence of function-blocking mAbs against CD14, TLR1, TLR2, TLR4, and TLR6. These receptors are expressed on cell surface of both human monocytes and monocytic cell line THP-1 (29, 53, 54). The pretreatment of THP-1 cells with MY4, an Ab blocking the mCD14-part of the LPS receptor (55), led to significant inhibition (p < 0.05) of rMip-mediated TNF-{alpha} production, indicating that rMip binds to mCD14 at the LPS-binding site as already observed for spirochaetal lipoproteins (53, 56, 57). The pretreatment of THP-1 cells with TL2.1, a specific blocker of extracellular human TLR2 (58) or GD2.F4, a mAb blocking TLR1 (59), led to significant inhibitions (p < 0.05) of rMip-mediated TNF-{alpha} production, whereas no significant effect was observed neither in presence of HTA125, an anti-TLR4 blocking mAb (60, 61) nor in presence of the clone 127, an anti-TLR6 blocking mAb (29). The combination of anti-TLR2 with either anti-TLR6 or -CD14 led to clear inhibitions (p < 0.005), and the combination of anti-TLR2 with anti-TLR1 to an almost complete inhibition (p < 0.0001) of TNF-{alpha} secretion, as shown in Fig. 11A. When inactivated C. trachomatis EB were used as stimulant, the combination of anti-TLR2 with anti-CD14, anti-TLR1, or anti-TLR6 induced significant inhibitions (p < 0.05 to 0.005), whereas the combination of anti-TLR4 with anti-CD14 provoked no significant reduction of EB-mediated TNF-{alpha} synthesis, as shown in Fig. 11B. Because TLR1 and TLR2 have been shown to be required for recognition of triacylated lipopeptides, such as Pam3CSK4 (62, 63, 64) whereas TLR6 and TLR2 were required for recognition of diacylated lipopeptides such as Pam2CSK4 but not for recognition of triacylated lipopeptides (63, 65), these two prototypic synthetic lipopeptides were used as controls and tested in the same conditions. When Pam3CSK4 was used as stimulant, the pretreatment of THP-1 cells with the combination of anti-TLR2 with anti-CD14, anti-TLR1, or anti-TLR6 led to significant inhibitions (p < 0.05 to 0.0001) of TNF-{alpha} secretion, the association of anti-TLR2 with anti-TLR1 being the most inhibitory, as shown in Fig. 12A. When Pam2CSK4 was used as stimulant, the pretreatment of THP-1 cells with the combination of anti-TLR2 with anti-TLR1 or anti-TLR6 led to significant inhibitions (p < 0.05 to 0.0001) of TNF-{alpha} secretion, the association of anti-TLR2 with anti-TLR6 being the most inhibitory, as shown in Fig. 12B. These data show no clear-cut segregation of Pam3CSK4 and Pam2CSK4 interactions with TLR2/TLR1 or TLR2/TLR6 (62, 63, 64, 65) but rather a tendency of Pam3CSK4 to stimulate THP-1 cells more efficiently via TLR2/TLR1 and Pam2CSK4 via TLR2/TLR6. Because E. coli LPS is commonly used as TLR4 and CD14-ligand (66), it was also used as control and tested in the same conditions. The pretreatment of THP-1 cells with the combination of anti-TLR4 with anti-CD14 led to significant inhibitions of TNF-{alpha} secretion (p < 0.005), whereas no inhibition was obtained with the combination of anti-TLR2 with anti-CD14, as shown in Fig. 12C. In all these experiments, no significant change of cytokine production was induced by nonimmune mouse IgG used as controls instead of each mAb.


Figure 11
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  		FIGURE 11. The production of TNF-{alpha} by PMA-differentiated THP-1 cells upon rMip or inactivated C. trachomatis EB activation is mainly TLR2/TLR1/TLR6/CD14 dependent. PMA-differentiated THP-1 cells were preincubated for 1 h with 50 µg/ml human IgG to block Fc receptors and prevent nonspecific binding of blocking Ab or control IgG. Cells were next preincubated for 1 h at 37°C in the presence or absence of 5 µg/ml of various blocking mAbs: anti-TLR1, anti-TLR2, anti-TLR4, anti-TLR6, anti-CD14, or isotype-matched mouse IgG1, IgG2a, and IgG2b controls alone or associated before addition of either 1 µg/ml rMip (A) or inactivated C. trachomatis EB (5 x 106/ml) (B). Results were expressed as TNF-{alpha} concentrations obtained in each condition, included pretreatment of cells with isotype controls. Each value represents mean ± SD of triplicates from one representative of two or three independent experiments with similar results. *, p < 0.05; **, p < 0.005; ***, p < 0.0001 determined by comparison with control using Student’s t test.

 

Figure 12
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  		FIGURE 12. Host receptors involved in activation of THP-1 cells by control lipopeptides and E. coli LPS. PMA-differentiated THP-1 cells were preincubated for 1 h with 50 µg/ml human IgG to block Fc receptors and prevent nonspecific binding of blocking Ab or control IgG. Cells were next preincubated for 1 h at 37°C in the presence or absence of 5 µg/ml of various association of blocking mAbs: anti-TLR1/TLR2, anti-TLR2/TLR6, anti-TLR2/CD14, anti-TLR4/CD14, or isotype-matched mouse IgG before addition of 0.01 µg/ml lipopeptides (Pam3CSK4 (A) or Pam2CSK4 (B)) or 1 µg/ml E. coli LPS (C). Results were expressed as TNF-{alpha} concentrations obtained in each condition, included pretreatment of cells with isotype controls. Each value represents mean ± SD of triplicates from one representative of two or three independent experiments with similar results. *, p < 0.05; **, p < 0.005; ***, p < 0.0001 were determined by comparison with control using Student’s t test.

 
CD14, TLR1, TLR2, and TLR6 are involved in rMip activation of HEK-293 cell lines expressing human TLR1/2, TLR2/6, or TLR2/CD14

To further ensure proper identification of receptors involved in the recognition of rMip and to assess their respective contribution, a cell model using HEK-293 cells transfected with human TLR1/2, TLR2/6, or TLR2/CD14 genes was used. Null cells lacking TLRs and CD14 responded to rMip by producing a 2.8-fold increase of IL-8 release in absence of stimulation, indicating that rMip was able to slightly stimulate these cells in absence of TLRs and CD14 but no response was observed with other stimulants. In contrast, HEK-293 cells expressing either hTLR1/2, or hTLR2/6, or hTLR2/CD14 responded to all stimulants, except E. coli LPS. The highest stimulatory effect of rMip was obtained under the condition of hTLR1/2 coexpression that led to a 26-fold higher release of IL-8 than in absence of stimulant (p < 0.0001). The hTLR2/6 and hTLR2/CD14 coexpressions led to a 16- and 9-fold increase of IL-8 release (p < 0.005), respectively. In presence of inactivated C. trachomatis EB, the highest stimulatory effect was also obtained under the condition of hTLR1/2 coexpression that led to a 6-fold higher release of IL-8 than in absence of stimulant (p < 0.0001). The hTLR2/6 and hTLR2/CD14 coexpressions led to a 3- and 5-fold increase of IL-8 release (p < 0.05 to 0.005), respectively. These results agree with those obtained in blocking experiments. In presence of lipopeptides, the effects were dependent upon the stimulating concentrations. At 1 µg/ml concentration, HEK-293 cells coexpressing hTLR1/2, hTLR2/6, or hTLR2/CD14 responded to both Pam3CSK4 and Pam2CSK4. The highest stimulatory effect of Pam3CSK4 was obtained under the condition of hTLR1/2 coexpression that led to a 49-fold increase whereas the coexpression of hTLR2/6 led to a 15-fold increase of IL-8 release (p < 0.005). When hTLR1/2 genes were coexpressed, Pam2CSK4 stimulation led to a lower increase of IL-8 release than under Pam3CSK4 stimulation (26- vs 49-fold) but when hTLR2/6 genes were coexpressed, Pam2CSK4 stimulation led only to a slightly higher increase of IL-8 release than under Pam3CSK4 stimulation (20 vs 15-fold). In contrast, when the stimulating concentration was 0.01 µg/ml, Pam3CSK4 was still able to stimulate HEK-293 cells coexpressing hTLR1/2 that led to a 11-fold increase of IL-8 release (p < 0.05) but was no more able to stimulate cells coexpressing hTLR2/6. However, Pam2CSK4 was still able to stimulate HEK-293 cells coexpressing hTLR1/2 and coexpressing hTLR2/6 that led to a 9- and 5-fold increase of IL-8 release, respectively (p < 0.005), as shown in Fig. 13. These results agree with those obtained in blocking experiments except that blocking of both TLR2 and TLR6 slightly inhibited Pam3CSK4 activation and that blocking of both TLR2 and CD14 did not inhibit Pam2CSK activation. These discrepancies could be explained by different expression ratios of TLR6 and CD14 in HEK compared with THP-1 cells. Taken together, these data indicate that rMip, inactivated C. trachomatis EB, Pam3CSK4, and Pam2CSK4 are only partially TLR1-, TLR6-, and CD14-dependent. These results are therefore partially contradictory to previous reports identifying TLR1 as the sole coreceptor for triacylated lipopeptides (62), and TLR6 as the sole coreceptor for diacylated lipopeptides (67) while supporting results of other groups (68, 69, 70).


Figure 13
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  		FIGURE 13. The production of IL-8 by HEK-293 cell lines expressing human TLR1/2, TLR2/6, or TLR2/CD14 upon rMip or inactivated C. trachomatis EB activation is mainly TLR2/TLR1/TLR6/CD14 dependent. Null, hTLR1/2, hTLR2/6, and hTLR2/CD14 cells were stimulated with 1 µg/ml rMip, inactivated C. trachomatis EB (5 x 106/ml), 1 or 0.01 µg/ml lipopeptides (Pam3CSK4 or Pam2CSK4), or 1 µg/ml E. coli LPS for 24 h. Culture supernatants were collected, and IL-8 content was analyzed. The results are expressed in terms of fold increase over the IL-8 levels of unstimulated cells. Each value represents mean ± SD of triplicates from three independent experiments. *, p < 0.05; **, p < 0.005; ***, p < 0.0001 determined by one sample t test; mean is significantly different from 1.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The present study demonstrates that among seven individual chlamydial components, rMip was the most effective to induce a proinflammatory cytokine response in human monocytes/macrophages (peripheral blood monocytes and THP-1 cell line). Even if rMip responses were lower than those induced by E. coli and S. typhimurium LPS, they were significant in contrast to Chlamydia LPS, as already reported (10, 15, 36, 71). IL-8 was the dominant cytokine induced by rMip in vitro and this observation must be related to the cytokine patterns present in vivo during infection by chlamydiae where high levels of IL-8 were reported in tears from children with trachoma, as well as in endocervical secretions obtained from women infected with Chlamydia (72).

The proinflammatory activity induced by WT rMip was not due to E. coli LPS contamination, as determined by the Limulus Amoebocyte lysate assay, insensitivity to polymyxin B, different serum requirement, and absence of inhibition by anti-TLR4 Abs. The stimulatory activities of WT rMip were similar to those of native Mip and appeared to be dependent upon lipid modification because nonlipidated C20A rMip variant was devoid of effect on cytokine release. In addition, WT rMip activity was greatly reduced by alkaline hydrolysis or treatment with lipases but was unaffected by heat or proteinase K treatments. The cell receptors involved in rMip and C. trachomatis EB cellular activation were determined using two independent assays that clearly demonstrated the involvement of TLR1/2/6 and CD14 but not TLR4. The main receptors involved in rMip activation were TLR1/2 but the co-presence of TLR1, TLR6, and CD14 was not absolutely required because activation was possible by TLR1/2, TLR2/6, or TLR2/CD14. In these assays, the receptors involved in Pam3CSK4 and Pam2CSK4 activation did not completely agree with the original concept, according to which triacylated lipopeptides recognize TLR2/TLR1 heteromers, whereas diacylated lipopeptides recognize TLR2/TLR6 heteromers (63, 65, 73) but agree with other reports (68, 69, 70). Indeed, the ability of Pam3CSK4 to stimulate HEK-293 cells coexpressing hTLR1/2 and, to a lesser extent, hTLR2/6 has been reported (68) and Pam3CSK4 has been shown to exhibit some activity toward TLR2/6 when used at high concentrations (69). Pam2CSK4 has been shown to exhibit comparable activities toward both the human TLR2/1 and TLR2/6 pairs (69) and macrophages of TLR6-deficient mice to be fully responsive to Pam2CSK4 (70). In fact the number of acyl-residues, the peptide sequence and the whole molecular structure of the lipopeptide/lipoprotein have been shown to be responsible for TLR2/1- or TLR2/6-dependent signaling (70, 74). The involvement of CD14 in rMip activation agrees with other studies showing that bacterial lipoproteins (53, 56, 57, 75, 76), Pam3CSK4 (53, 77), and Pam2CSK4 (68) interact with CD14 to cause cytokine induction. These results agree with reports showing a predominant role of TLR2 in C. trachomatis (78) as well as C. pneumoniae recognition (14, 19, 79) and with results of Netea et al. (14) attributing proinflammatory cytokine production to non-LPS components. The fact that rMip and EB act through the same receptors and that anti-rMip Ab is able to partly inhibit EB-mediated TNF-{alpha} release suggest a role of native Mip, present at the EB surface (20), or other surface lipoproteins in EB recognition and macrophage activation in natural infection. If IL-1β, TNF-{alpha}, and IL-6 release in response to native Mip or other lipoproteins may aid in eradicating Chlamydia infection (80, 81, 82, 83), particularly if their levels increase early (83), these cytokines may also promote long-term tissue damage. As an example, high levels of TNF-{alpha} have been associated with detrimental effects in ocular chlamydial infection (84). Moreover, Mip-induced IL-8 can be deleterious (83), particularly in case of chronic secretion. Indeed, IL-8 promotes the infiltration of neutrophils that are not only inefficient in resolving chlamydial infections but can release proteases that damage cells. In addition and as reported for other bacterium-associated lipoproteins (85), Mip or other lipoproteins could be released from EB surface (either from living bacteria or from bacteria lysed as a result of effective host defense or the activities of certain antibiotics) and retained inside tissues where they might activate resident cells and perpetuate inflammation even after the eradication of live bacteria with antibiotic therapy. Lipoproteins are considered as crucial virulence factors in inflammatory processes and in pathogenesis of several important bacterial infections, such as those triggered by Mycobacterium tuberculosis (76), Neisseria gonorrhoeae (86), Listeria monocytogenes (58, 87), Brucella abortus (88), and members of Enterobacteriaceae family (89, 90). In organisms such as Borrelia burgdorferi and Treponema pallidum, which lack LPS, bacterial lipoproteins are known to play an important role in pathogenesis (49, 75, 88, 91, 92, 93). In chlamydiae, LPS is a major structural component of all chlamydial species (22) but compared with the LPS of enterobacteria, it has much lower endotoxin activity (10, 71, 94, 95) because its lipid A is highly hydrophobic and has unique structural features with the presence of unusual, long-chain fatty acids (96). Chlamydia LPS is unable to elicit inflammation in experimental animals (13) and is a weak inducer of the inflammatory cytokine response (10, 36, 37), as shown in the present study. All these data support the hypothesis that in chlamydiae, Mip or other lipoproteins might play a key role in pathogenesis. The fact that Mip is present in the membrane of EBs as well as reticulate bodies (97) and was recently shown to be surface exposed by two different approaches (20) reinforces its potential role in pathogenesis of Chlamydia infection. If the exact chemical nature of Chlamydia-derived monocyte/macrophage stimulators is not known, the fact that receptors involved in Mip recognition are similar to those involved in C. trachomatis EB recognition is supportive of the involvement of Mip or other Chlamydia-associated lipoproteins as inflammatory active element of EB.

In conclusion, this study is the first report about a chlamydial lipoprotein displaying proinflammatory properties. As Mip appears to be present in different species of the Chlamydiaceae family (20), it could have an important role in the inflammatory aspects of trachoma, reactive arthritis, or atherogenesis.


   Acknowledgment
 
We thank Yvette Froment for technical support.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from Novartis, Albert-Boeni, and de Reuter Foundations, and from Geneva Academic Society. This work was also supported by Grants 3200B0-107883 (to S.B.) and 3200-107592/1 (to C.G.) from the Swiss National Science Foundation. Back

2 Address correspondence and reprint requests to Dr. Sylvette Bas, Division of Rheumatology, Department of Internal Medicine, University Hospital, 1211 Geneva 14, Switzerland. E-mail address: sylvette.bas{at}hcuge.ch Back

3 Abbreviations used in this paper: EB, elementary body; hsp, heat shock protein; pgp3, polypeptide encoded by open reading frame 3 of the plasmid; Mip, macrophage infectivity potentiator; Omp2, outer membrane protein 2; WT, wild type; HEK, human embryonic kidney. Back

Received for publication May 31, 2007. Accepted for publication November 12, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Hahn, D. L., R. W. Dodge, R. Golubjatnikov. 1991. Association of Chlamydia pneumoniae (strain TWAR) infection with wheezing, asthmatic bronchitis, and adult-onset asthma. J. Am. Med. Assoc. 266: 225-230. [Abstract/Free Full Text]
  2. Centers for Disease Control and Prevention 1997. Chlamydia trachomatis genital infections–United States, 1995. J. Am. Med. Assoc. 277: 952-953. [Abstract/Free Full Text]
  3. Kalayoglu, M. V., P. Libby, G. I. Byrne. 2002. Chlamydia pneumoniae as an emerging risk factor in cardiovascular disease. J. Am. Med. Assoc. 288: 2724-2731. [Abstract/Free Full Text]
  4. Mabey, D. C., A. W. Solomon, A. Foster. 2003. Trachoma. Lancet 362: 223-229. [Medline]
  5. Corsaro, D., G. Greub. 2006. Pathogenic potential of novel Chlamydiae and diagnostic approaches to infections due to these obligate intracellular bacteria. Clin. Microbiol. Rev. 19: 283-297. [Abstract/Free Full Text]
  6. Patton, D. L., C. C. Kuo. 1989. Histopathology of Chlamydia trachomatis salpingitis after primary and repeated reinfections in the monkey subcutaneous pocket model. J. Reprod Fertil. 85: 647-656. [Abstract/Free Full Text]
  7. Kiviat, N. B., J. A. Paavonen, P. Wolner-Hanssen, C. W. Critchlow, W. E. Stamm, J. Douglas, D. A. Eschenbach, L. A. Corey, K. K. Holmes. 1990. Histopathology of endocervical infection caused by Chlamydia trachomatis, herpes simplex virus, Trichomonas vaginalis, and Neisseria gonorrhoeae. Hum. Pathol. 21: 831-837. [Medline]
  8. Morrison, R. P., R. J. Belland, K. Lyng, H. D. Caldwell. 1989. Chlamydial disease pathogenesis. The 57-kD chlamydial hypersensitivity antigen is a stress response protein. J. Exp. Med. 170: 1271-1283. [Abstract/Free Full Text]
  9. Rothermel, C. D., J. Schachter, P. Lavrich, E. C. Lipsitz, T. Francus. 1989. Chlamydia trachomatis-induced production of interleukin-1 by human monocytes. Infect. Immun. 57: 2705-2711. [Abstract/Free Full Text]
  10. Ingalls, R. R., P. A. Rice, N. Qureshi, K. Takayama, J. S. Lin, D. T. Golenbock. 1995. The inflammatory cytokine response to Chlamydia trachomatis infection is endotoxin mediated. Infect. Immun. 63: 3125-3130. [Abstract]
  11. Kol, A., G. K. Sukhova, A. H. Lichtman, P. Libby. 1998. Chlamydial heat shock protein 60 localizes in human atheroma and regulates macrophage tumor necrosis factor-{alpha} and matrix metalloproteinase expression. Circulation 98: 300-307. [Abstract/Free Full Text]
  12. Kol, A., T. Bourcier, A. H. Lichtman, P. Libby. 1999. Chlamydial and human heat shock protein 60s activate human vascular endothelium, smooth muscle cells, and macrophages. J. Clin. Invest. 103: 571-577. [Medline]
  13. Watkins, N. G., W. J. Hadlow, A. B. Moos, H. D. Caldwell. 1986. Ocular delayed hypersensitivity: a pathogenetic mechanism of chlamydial-conjunctivitis in guinea pigs. Proc. Natl. Acad. Sci. USA 83: 7480-7484. [Abstract/Free Full Text]
  14. Netea, M. G., B. J. Kullberg, J. M. Galama, A. F. Stalenhoef, C. A. Dinarello, J. W. Van der Meer. 2002. Non-LPS components of Chlamydia pneumoniae stimulate cytokine production through Toll-like receptor 2-dependent pathways. Eur. J. Immunol. 32: 1188-1195. [Medline]
  15. Prebeck, S., H. Brade, C. J. Kirschning, C. P. da Costa, S. Durr, H. Wagner, T. Miethke. 2003. The Gram-negative bacterium Chlamydia trachomatis L2 stimulates tumor necrosis factor secretion by innate immune cells independently of its endotoxin. Microbes Infect. 5: 463-470. [Medline]
  16. Vabulas, R. M., P. Ahmad-Nejad, C. da Costa, T. Miethke, C. J. Kirschning, H. Hacker, H. Wagner. 2001. Endocytosed HSP60s use toll-like receptor 2 (TLR2) and TLR4 to activate the toll/interleukin-1 receptor signaling pathway in innate immune cells. J. Biol. Chem. 276: 31332-31339. [Abstract/Free Full Text]
  17. Sasu, S., D. LaVerda, N. Qureshi, D. T. Golenbock, D. Beasley. 2001. Chlamydia pneumoniae and chlamydial heat shock protein 60 stimulate proliferation of human vascular smooth muscle cells via toll-like receptor 4 and p44/p42 mitogen-activated protein kinase activation. Circ. Res. 89: 244-250. [Abstract/Free Full Text]
  18. Bulut, Y., E. Faure, L. Thomas, H. Karahashi, K. S. Michelsen, O. Equils, S. G. Morrison, R. P. Morrison, M. Arditi. 2002. Chlamydial heat shock protein 60 activates macrophages and endothelial cells through Toll-like receptor 4 and MD2 in a MyD88-dependent pathway. J. Immunol. 168: 1435-1440. [Abstract/Free Full Text]
  19. Prebeck, S., C. Kirschning, S. Durr, C. da Costa, B. Donath, K. Brand, V. Redecke, H. Wagner, T. Miethke. 2001. Predominant role of toll-like receptor 2 versus 4 in Chlamydia pneumoniae-induced activation of dendritic cells. J. Immunol. 167: 3316-3323. [Abstract/Free Full Text]
  20. Neff, L., S. Daher, P. Muzzin, U. Spenato, F. Gülacar, C. Gabay, S. Bas. 2007. Molecular characterization and subcellular localization of macrophage infectivity potentiator, a Chlamydia trachomatis lipoprotein. J. Bacteriol. 189: 4739-4748. [Abstract/Free Full Text]
  21. Boleti, H., A. Benmerah, D. M. Ojcius, N. Cerf-Bensussan, A. Dautry-Varsat. 1999. Chlamydia infection of epithelial cells expressing dynamin and Eps15 mutants: clathrin-independent entry into cells and dynamin-dependent productive growth. J. Cell Sci. 112: (Pt. 10):1487-1496. [Abstract]
  22. Rund, S., B. Lindner, H. Brade, O. Holst. 1999. Structural analysis of the lipopolysaccharide from Chlamydia trachomatis serotype L2. J. Biol. Chem. 274: 16819-16824. [Abstract/Free Full Text]
  23. Hirschfeld, M., Y. Ma, J. H. Weis, S. N. Vogel, J. J. Weis. 2000. Cutting edge: repurification of lipopolysaccharide eliminates signaling through both human and murine toll-like receptor 2. J. Immunol. 165: 618-622. [Abstract/Free Full Text]
  24. Bas, S., P. Muzzin, B. Ninet, J. E. Bornand, C. Scieux, T. L. Vischer. 2001. Chlamydial serology: comparative diagnostic value of immunoblotting, microimmunofluorescence test, and immunoassays using different recombinant proteins as antigens. J. Clin. Microbiol. 39: 1368-1377. [Abstract/Free Full Text]
  25. Bas, S., P. Muzzin, T. L. Vischer. 2001. Chlamydia trachomatis serology: diagnostic value of outer membrane protein 2 compared with that of other antigens. J. Clin. Microbiol. 39: 4082-4085. [Abstract/Free Full Text]
  26. Bausinger, H., D. Lipsker, U. Ziylan, S. Manié, J. P. Briand, J. P. Cazenave, S. Muller, J. F. Haeuw, C. Ravanat, H. de la Salle, D. Hanau. 2002. Endotoxin-free heat-shock protein 70 fails to induce APC activation. Eur. J. Immunol. 32: 3708-3713. [Medline]
  27. Gao, B., M. F. Tsan. 2003. Recombinant human heat shock protein 60 does not induce the release of tumor necrosis factor {alpha} from murine macrophages. J. Biol. Chem. 278: 22523-22529. [Abstract/Free Full Text]
  28. Gao, B., M. F. Tsan. 2003. Endotoxin contamination in recombinant human heat shock protein 70 (Hsp70) preparation is responsible for the induction of tumor necrosis factor {alpha} release by murine macrophages. J. Biol. Chem. 278: 174-179. [Abstract/Free Full Text]
  29. Nakao, Y., K. Funami, S. Kikkawa, M. Taniguchi, M. Nishiguchi, Y. Fukumori, T. Seya, M. Matsumoto. 2005. Surface-expressed TLR6 participates in the recognition of diacylated lipopeptide and peptidoglycan in human cells. J. Immunol. 174: 1566-1573. [Abstract/Free Full Text]
  30. Armant, M., M. Rubio, G. Delespesse, M. Sarfati. 1995. Soluble CD23 directly activates monocytes to contribute to the antigen-independent stimulation of resting T cells. J. Immunol. 155: 4868-4875. [Abstract]
  31. Burger, D., N. Molnarfi, L. Gruaz, J. M. Dayer. 2004. Differential induction of IL-1β and TNF by CD40 ligand or cellular contact with stimulated T cells depends on the maturation stage of human monocytes. J. Immunol. 173: 1292-1297. [Abstract/Free Full Text]
  32. Tsuchiya, S., M. Yamabe, Y. Yamaguchi, Y. Kobayashi, T. Konno, K. Tada. 1980. Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int. J. Cancer 26: 171-176. [Medline]
  33. Muhlradt, P. F., M. Frisch. 1994. Purification and partial biochemical characterization of a Mycoplasma fermentans-derived substance that activates macrophages to release nitric oxide, tumor necrosis factor, and interleukin-6. Infect. Immun. 62: 3801-3807. [Abstract/Free Full Text]
  34. Pleiss, J., M. Fischer, R. D. Schmid. 1998. Anatomy of lipase binding sites: the scissile fatty acid binding site. Chem. Phys. Lipids 93: 67-80. [Medline]
  35. Vidal, V., I. G. Scragg, S. J. Cutler, K. A. Rockett, D. Fekade, D. A. Warrell, D. J. Wright, D. Kwiatkowski. 1998. Variable major lipoprotein is a principal TNF-inducing factor of louse-borne relapsing fever. Nat. Med. 4: 1416-1420. [Medline]
  36. Brade, L., S. Schramek, U. Schade, H. Brade. 1986. Chemical, biological, and immunochemical properties of the Chlamydia psittaci lipopolysaccharide. Infect. Immun. 54: 568-574. [Abstract/Free Full Text]
  37. Karimi, S. T., R. H. Schloemer, C. E. Wilde, III. 1989. Accumulation of chlamydial lipopolysaccharide antigen in the plasma membranes of infected cells. Infect. Immun. 57: 1780-1785. [Abstract/Free Full Text]
  38. Häupl, T., S. Landgraf, P. Netusil, N. Biller, C. Capiau, P. Desmons, P. Hauser, G. R. Burmester. 1997. Activation of monocytes by three OspA vaccine candidates: lipoprotein OspA is a potent stimulator of monokines. FEMS Immunol. Med. Microbiol. 19: 15-23. [Medline]
  39. Murthy, P. K., V. A. Dennis, B. L. Lasater, M. T. Philipp. 2000. Interleukin-10 modulates proinflammatory cytokines in the human monocytic cell line THP-1 stimulated with Borrelia burgdorferi lipoproteins. Infect. Immun. 68: 6663-6669. [Abstract/Free Full Text]
  40. Auwerx, J.. 1991. The human leukemia cell line, THP-1: a multifacetted model for the study of monocyte-macrophage differentiation. Experientia 47: 22-31. [Medline]
  41. Schumann, R. R., S. R. Leong, G. W. Flaggs, P. W. Gray, S. D. Wright, J. C. Mathison, P. S. Tobias, R. J. Ulevitch. 1990. Structure and function of lipopolysaccharide binding protein. Science 249: 1429-1431. [Abstract/Free Full Text]
  42. Wright, S. D., R. A. Ramos, P. S. Tobias, R. J. Ulevitch, J. C. Mathison. 1990. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science 249: 1431-1433. [Abstract/Free Full Text]
  43. Corradin, S. B., J. Mauel, P. Gallay, D. Heumann, R. J. Ulevitch, P. S. Tobias. 1992. Enhancement of murine macrophage binding of and response to bacterial lipopolysaccharide (LPS) by LPS-binding protein. J. Leukocyte Biol. 52: 363-368. [Medline]
  44. Hailman, E., H. S. Lichenstein, M. M. Wurfel, D. S. Miller, D. A. Johnson, M. Kelley, L. A. Busse, M. M. Zukowski, S. D. Wright. 1994. Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14. J. Exp. Med. 179: 269-277. [Abstract/Free Full Text]
  45. Lundemose, A. G., D. A. Rouch, S. Birkelund, G. Christiansen, J. H. Pearce. 1992. Chlamydia trachomatis Mip-like protein. Mol. Microbiol. 6: 2539-2548. [Medline]
  46. Muhlradt, P. F., H. Meyer, R. Jansen. 1996. Identification of S-(2,3-dihydroxypropyl)cystein in a macrophage-activating lipopeptide from Mycoplasma fermentans. Biochemistry 35: 7781-7786. [Medline]
  47. Brandt, M. E., B. S. Riley, J. D. Radolf, M. V. Norgard. 1990. Immunogenic integral membrane proteins of Borrelia burgdorferi are lipoproteins. Infect. Immun. 58: 983-991. [Abstract/Free Full Text]
  48. Shimizu, T., Y. Iwamoto, Y. Yanagihara, M. Kurimura, K. Achiwa. 1994. Mitogenic activity and the induction of tumor necrosis factor by lipopeptide analogs of the N-terminal part of lipoprotein in the outer membrane of Escherichia coli. Biol. Pharm. Bull. 17: 980-982. [Medline]
  49. Radolf, J. D., L. L. Arndt, D. R. Akins, L. L. Curetty, M. E. Levi, Y. Shen, L. S. Davis, M. V. Norgard. 1995. Treponema pallidum and Borrelia burgdorferi lipoproteins and synthetic lipopeptides activate monocytes/macrophages. J. Immunol. 154: 2866-2877. [Abstract]
  50. Metzger, J. W., A. G. Beck-Sickinger, M. Loleit, M. Eckert, W. G. Bessler, G. Jung. 1995. Synthetic S-(2,3-dihydroxypropyl)-cysteinyl peptides derived from the N-terminus of the cytochrome subunit of the photoreaction centre of Rhodopseudomonas viridis enhance murine splenocyte proliferation. J. Pept. Sci. 1: 184-190. [Medline]
  51. Muhlradt, P. F., M. Kiess, H. Meyer, R. Sussmuth, G. Jung. 1997. Isolation, structure elucidation, and synthesis of a macrophage stimulatory lipopeptide from Mycoplasma fermentans acting at picomolar concentration. J. Exp. Med. 185: 1951-1958. [Abstract/Free Full Text]
  52. Janeway, C. A., Jr, R. Medzhitov. 2002. Innate immune recognition. Annu. Rev. Immunol. 20: 197-216. [Medline]
  53. Sellati, T. J., D. A. Bouis, R. L. Kitchens, R. P. Darveau, J. Pugin, R. J. Ulevitch, S. C. Gangloff, S. M. Goyert, M. V. Norgard, J. D. Radolf. 1998. Treponema pallidum and Borrelia burgdorferi lipoproteins and synthetic lipopeptides activate monocytic cells via a CD14-dependent pathway distinct from that used by lipopolysaccharide. J. Immunol. 160: 5455-5464. [Abstract/Free Full Text]
  54. Visintin, A., A. Mazzoni, J. H. Spitzer, D. H. Wyllie, S. K. Dower, D. M. Segal. 2001. Regulation of Toll-like receptors in human monocytes and dendritic cells. J. Immunol. 166: 249-255. [Abstract/Free Full Text]
  55. Triantafilou, M., K. Triantafilou, N. Fernandez. 2000. Rough and smooth forms of fluorescein-labelled bacterial endotoxin exhibit CD14/LBP dependent and independent binding that is influenced by endotoxin concentration. Eur. J. Biochem. 267: 2218-2226. [Medline]
  56. Wooten, R. M., T. B. Morrison, J. H. Weis, S. D. Wright, R. Thieringer, J. J. Weis. 1998. The role of CD14 in signaling mediated by outer membrane lipoproteins of Borrelia burgdorferi. J. Immunol. 160: 5485-5492. [Abstract/Free Full Text]
  57. Giambartolomei, G. H., V. A. Dennis, B. L. Lasater, M. T. Philipp. 1999. Induction of pro- and anti-inflammatory cytokines by Borrelia burgdorferi lipoproteins in monocytes is mediated by CD14. Infect. Immun. 67: 140-147. [Abstract/Free Full Text]
  58. Flo, T. H., O. Halaas, E. Lien, L. Ryan, G. Teti, D. T. Golenbock, A. Sundan, T. Espevik. 2000. Human toll-like receptor 2 mediates monocyte activation by Listeria monocytogenes, but not by group B streptococci or lipopolysaccharide. J. Immunol. 164: 2064-2069. [Abstract/Free Full Text]
  59. Wyllie, D. H., E. Kiss-Toth, A. Visintin, S. C. Smith, S. Boussouf, D. M. Segal, G. W. Duff, S. K. Dower. 2000. Evidence for an accessory protein function for Toll-like receptor 1 in anti-bacterial responses. J. Immunol. 165: 7125-7132. [Abstract/Free Full Text]
  60. Shimazu, R., S. Akashi, H. Ogata, Y. Nagai, K. Fukudome, K. Miyake, M. Kimoto. 1999. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J. Exp. Med. 189: 1777-1782. [Abstract/Free Full Text]
  61. Akashi, S., H. Ogata, F. Kirikae, T. Kirikae, K. Kawasaki, M. Nishijima, R. Shimazu, Y. Nagai, K. Fukudome, M. Kimoto, K. Miyake. 2000. Regulatory roles for CD14 and phosphatidylinositol in the signaling via toll-like receptor 4-MD-2. Biochem. Biophys. Res. Commun. 268: 172-177. [Medline]
  62. Takeuchi, O., S. Sato, T. Horiuchi, K. Hoshino, K. Takeda, Z. Dong, R. L. Modlin, S. Akira. 2002. Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J. Immunol. 169: 10-14. [Abstract/Free Full Text]
  63. Akira, S.. 2003. Mammalian Toll-like receptors. Curr. Opin. Immunol. 15: 5-11. [Medline]
  64. Sandor, F., E. Latz, F. Re, L. Mandell, G. Repik, D. T. Golenbock, T. Espevik, E. A. Kurt-Jones, R. W. Finberg. 2003. Importance of extra- and intracellular domains of TLR1 and TLR2 in NF{kappa}B signaling. J. Cell Biol. 162: 1099-1110. [Abstract/Free Full Text]
  65. Takeuchi, O., T. Kawai, P. F. Muhlradt, M. Morr, J. D. Radolf, A. Zychlinsky, K. Takeda, S. Akira. 2001. Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int. Immunol. 13: 933-940. [Abstract/Free Full Text]
  66. Takeda, K., T. Kaisho, S. Akira. 2003. Toll-like receptors. Annu. Rev. Immunol. 21: 335-376. [Medline]
  67. Morr, M., O. Takeuchi, S. Akira, M. M. Simon, P. F. Muhlradt. 2002. Differential recognition of structural details of bacterial lipopeptides by toll-like receptors. Eur. J. Immunol. 32: 3337-3347. [Medline]
  68. Schroder, N. W., H. Heine, C. Alexander, M. Manukyan, J. Eckert, L. Hamann, U. B. Gobel, R. R. Schumann. 2004. Lipopolysaccharide binding protein binds to triacylated and diacylated lipopeptides and mediates innate immune responses. J. Immunol. 173: 2683-2691. [Abstract/Free Full Text]
  69. Omueti, K. O., J. M. Beyer, C. M. Johnson, E. A. Lyle, R. I. Tapping. 2005. Domain exchange between human toll-like receptors 1 and 6 reveals a region required for lipopeptide discrimination. J. Biol. Chem. 280: 36616-36625. [Abstract/Free Full Text]
  70. Buwitt-Beckmann, U., H. Heine, K. H. Wiesmuller, G. Jung, R. Brock, S. Akira, A. J. Ulmer. 2005. Toll-like receptor 6-independent signaling by diacylated lipopeptides. Eur. J. Immunol. 35: 282-289. [Medline]
  71. Heine, H., S. Müller-Loennies, L. Brade, B. Lindner, H. Brade. 2003. Endotoxic activity and chemical structure of lipopolysaccharides from Chlamydia trachomatis serotypes E and L2 and Chlamydophila psittaci 6BC. Eur. J. Biochem. 270: 440-450. [Medline]
  72. Stephens, R. S.. 2003. The cellular paradigm of chlamydial pathogenesis. Trends Microbiol. 11: 44-51. [Medline]
  73. Takeda, K., O. Takeuchi, S. Akira. 2002. Recognition of lipopeptides by Toll-like receptors. J. Endotoxin Res. 8: 459-463. [Medline]
  74. Okusawa, T., M. Fujita, J. Nakamura, T. Into, M. Yasuda, A. Yoshimura, Y. Hara, A. Hasebe, D. T. Golenbock, M. Morita, et al 2004. Relationship between structures and biological activities of mycoplasmal diacylated lipopeptides and their recognition by toll-like receptors 2 and 6. Infect. Immun. 72: 1657-1665. [Abstract/Free Full Text]
  75. Hirschfeld, M., C. J. Kirschning, R. Schwandner, H. Wesche, J. H. Weis, R. M. Wooten, J. J. Weis. 1999. Cutting edge: inflammatory signaling by Borrelia burgdorferi lipoproteins is mediated by toll-like receptor 2. J. Immunol. 163: 2382-2386. [Abstract/Free Full Text]
  76. Brightbill, H. D., D. H. Libraty, S. R. Krutzik, R. B. Yang, J. T. Belisle, J. R. Bleharski, M. Maitland, M. V. Norgard, S. E. Plevy, S. T. Smale, et al 1999. Host defense mechanisms triggered by microbial lipoproteins through toll-like receptors. Science 285: 732-736. [Abstract/Free Full Text]
  77. Manukyan, M., K. Triantafilou, M. Triantafilou, A. Mackie, N. Nilsen, T. Espevik, K. H. Wiesmuller, A. J. Ulmer, H. Heine. 2005. Binding of lipopeptide to CD14 induces physical proximity of CD14, TLR2 and TLR1. Eur. J. Immunol. 35: 911-921. [Medline]
  78. Darville, T., J. M. O’Neill, C. W. Andrews, Jr, U. M. Nagarajan, L. Stahl, D. M. Ojcius. 2003. Toll-like receptor-2, but not Toll-like receptor-4, is essential for development of oviduct pathology in chlamydial genital tract infection. J. Immunol. 171: 6187-6197. [Abstract/Free Full Text]
  79. Netea, M. G., J. W. van der Meer, M. van Deuren, B. J. Kullberg. 2003. Proinflammatory cytokines and sepsis syndrome: not enough, or too much of a good thing?. Trends Immunol. 24: 254-258. [Medline]
  80. Williams, D. M., D. M. Magee, L. F. Bonewald, J. G. Smith, C. A. Bleicker, G. I. Byrne, J. Schachter. 1990. A role in vivo for tumor necrosis factor {alpha} in host defense against Chlamydia trachomatis. Infect. Immun. 58: 1572-1576. [Abstract/Free Full Text]
  81. Darville, T., C. W. Andrews, Jr, K. K. Laffoon, W. Shymasani, L. R. Kishen, R. G. Rank. 1997. Mouse strain-dependent variation in the course and outcome of chlamydial genital tract infection is associated with differences in host response. Infect. Immun. 65: 3065-3073. [Abstract]
  82. Williams, D. M., B. G. Grubbs, T. Darville, K. Kelly, R. G. Rank. 1998. A role for interleukin-6 in host defense against murine Chlamydia trachomatis infection. Infect. Immun. 66: 4564-4567. [Abstract/Free Full Text]
  83. Darville, T., C. W. Andrews, Jr, J. D. Sikes, P. L. Fraley, R. G. Rank. 2001. Early local cytokine profiles in strains of mice with different outcomes from chlamydial genital tract infection. Infect. Immun. 69: 3556-3561. [Abstract/Free Full Text]
  84. Conway, D. J., M. J. Holland, R. L. Bailey, A. E. Campbell, O. S. Mahdi, R. Jennings, E. Mbena, D. C. Mabey. 1997. Scarring trachoma is associated with polymorphism in the tumor necrosis factor {alpha} (TNF-{alpha}) gene promoter and with elevated TNF-{alpha} levels in tear fluid. Infect. Immun. 65: 1003-1006. [Abstract]
  85. Hellman, J., P. M. Loiselle, M. M. Tehan, J. E. Allaire, L. A. Boyle, J. T. Kurnick, D. M. Andrews, K. Sik Kim, H. S. Warren. 2000. Outer membrane protein A, peptidoglycan-associated lipoprotein, and murein lipoprotein are released by Escherichia coli bacteria into serum. Infect. Immun. 68: 2566-2572. [Abstract/Free Full Text]
  86. Fisette, P. L., S. Ram, J. M. Andersen, W. Guo, R. R. Ingalls. 2003. The Lip lipoprotein from Neisseria gonorrhoeae stimulates cytokine release and NF-{kappa}B activation in epithelial cells in a Toll-like receptor 2-dependent manner. J. Biol. Chem. 278: 46252-46260. [Abstract/Free Full Text]
  87. Réglier-Poupet, H., C. Frehel, I. Dubail, J. L. Beretti, P. Berche, A. Charbit, C. Raynaud. 2003. Maturation of lipoproteins by type II signal peptidase is required for phagosomal escape of Listeria monocytogenes. J. Biol. Chem. 278: 49469-49477. [Abstract/Free Full Text]
  88. Giambartolomei, G. H., A. Zwerdling, J. Cassataro, L. Bruno, C. A. Fossati, M. T. Philipp. 2004. Lipoproteins, not lipopolysaccharide, are the key mediators of the proinflammatory response elicited by heat-killed Brucella abortus. J. Immunol. 173: 4635-4642. [Abstract/Free Full Text]
  89. Zhang, H., J. W. Peterson, D. W. Niesel, G. R. Klimpel. 1997. Bacterial lipoprotein and lipopolysaccharide act synergistically to induce lethal shock and proinflammatory cytokine production. J. Immunol. 159: 4868-4878. [Abstract]
  90. Hellman, J., J. D. Roberts, Jr, M. M. Tehan, J. E. Allaire, H. S. Warren. 2002. Bacterial peptidoglycan-associated lipoprotein is released into the bloodstream in Gram-negative sepsis and causes inflammation and death in mice. J. Biol. Chem. 277: 14274-14280. [Abstract/Free Full Text]
  91. Infante-Duarte, C., T. Kamradt. 1997. Lipopeptides of Borrelia burgdorferi outer surface proteins induce Th1 phenotype development in alphabeta T-cell receptor transgenic mice. Infect. Immun. 65: 4094-4099. [Abstract]
  92. Porcella, S. F., T. G. Schwan. 2001. Borrelia burgdorferi and Treponema pallidum: a comparison of functional genomics, environmental adaptations, and pathogenic mechanisms. J. Clin. Invest. 107: 651-656. [Medline]
  93. Thoma-Uszynski, S., S. Stenger, O. Takeuchi, M. T. Ochoa, M. Engele, P. A. Sieling, P. F. Barnes, M. Rollinghoff, P. L. Bolcskei, M. Wagner, et al 2001. Induction of direct antimicrobial activity through mammalian toll-like receptors. Science 291: 1544-1547. [Abstract/Free Full Text]
  94. Nurminen, M., M. Leinonen, P. Saikku, P. H. Makela. 1983. The genus-specific antigen of Chlamydia: resemblance to the lipopolysaccharide of enteric bacteria. Science 220: 1279-1281. [Abstract/Free Full Text]
  95. Brade, H., L. Brade, F. E. Nano. 1987. Chemical and serological investigations on the genus-specific lipopolysaccharide epitope of Chlamydia. Proc. Natl. Acad. Sci. USA 84: 2508-2512. [Abstract/Free Full Text]
  96. Kosma, P.. 1999. Chlamydial lipopolysaccharide. Biochim. Biophys. Acta 1455: 387-402. [Medline]
  97. Lundemose, A. G., S. Birkelund, S. J. Fey, P. M. Larsen, G. Christiansen. 1991. Chlamydia trachomatis contains a protein similar to the Legionella pneumophila mip gene product. Mol. Microbiol. 5: 109-115. [Medline]



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