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International Immunology Advance Access originally published online on December 4, 2007
International Immunology 2008 20(2):199-208; doi:10.1093/intimm/dxm129
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Low potency of Chlamydophila LPS to activate human mononuclear cells due to its reduced affinities for CD14 and LPS-binding protein

Yuko Tsutsumi-Ishii1, Kazunori Shimada2, Hiroyuki Daida2, Rudolf Toman3 and Isao Nagaoka1

1 Department of Host Defense and Biochemical Research
2 Department of Cardiovascular Medicine, Juntendo University School of Medicine, Tokyo, Japan
3 Laboratory for Diagnosis and Prevention of Rickettsial and Chlamydial Infections, Institute of Virology, Slovak Academy of Science, Bratislava, Slovak Republic

Correspondence to: I. Nagaoka; E-mail: nagaokai{at}med.juntendo.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 References
 
Chlamydiaceae are small obligate intracellular parasites and classified as Gram-negative bacteria. Among Chlamydiaceae-derived components, LPS is known as an immunomodulator and possesses a unique lipid A structure with longer but fewer acyl chains. In this study, to elucidate the Chlamydiaceae-induced immune responses, we evaluated the actions of Chlamydophila psittaci LPS as a Chlamydiaceae LPS on human PBMCs and compared with those of Escherichia coli LPS. Similar to E. coli LPS, C. psittaci LPS bound to monocytes and induced the pro-inflammatory cytokine production in an LPS-binding protein (LBP)-dependent manner. However, C. psittaci LPS was much less potent than E. coli LPS in both the LPS binding and cytokine production. Interestingly, although the binding of C. psittaci LPS was mediated by CD14, Toll-like receptor 4 (TLR4) and CD11b, CD14 and TLR4 but not CD11b were involved in the cytokine production. Of note, ELISA-based binding assays revealed that C. psittaci LPS directly bound to LBP and CD14; however, the affinities were much less than those of E. coli LPS. Together, these observations possibly suggest that Chlamydiaceae LPS has low binding affinities for LPS recognition molecules such as CD14 and LBP and exhibit weak biological activities against host immune cells including monocytes, thereby contributing to the chronic (persistent) inflammatory reactions during infection.

Keywords: innate immunity, toll-like receptor


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
Chlamydiaceae are small obligate, intracellular, Gram-negative bacteria that cause various infectious diseases in humans and animals as common pathogens (1–3). Among Chlamydiaceae, Chlamydia trachomatis, Chlamydophila pneumoniae and Chlamydophila psittaci are pathogenic to humans and responsible for infectious diseases involving eyes, urogenital organs and respiratory tract (3, 4). The development of Chlamydiaceae-triggered diseases is closely associated with their persistent infection.

During infection, pathogens are eliminated by the combination of innate and acquired immunities. Chlamydiaceae, however, have evolved the strategies to escape from host defense by avoiding recognition as pathogens, inhibiting phagolysosome fusion and controlling host cell survival (5–7). Thus, Chlamydiaceae can survive in the infected cells and persistently activate host cells at the infection site. Although sustained infection is an important feature of Chlamydiaceae, little is known about the mechanisms for the host cell activation. Earlier studies reported that Chlamydiaceae-derived components such as LPS, major outer membrane protein (MOMP) and heat shock protein 60 (HSP60) as well as intact bacteria could activate macrophages and endothelial cells (7–10). Among these bacterial components, LPS is known as an immunomodulator, which induces pro-inflammatory cytokine production and mitogenic reaction (11–13). The biological activities of LPS are critically dependent on its lipid A portion, composed of phosphorylated glucosamine disaccharide and several acyl chains. Lipid A moieties of LPS from distinct bacteria species differ in the length, position and number of the acyl chains, and these variations reflect the bioactivities of lipid A (12–14). To date, structures of LPS from three species of Chlamydiaceae (C. pneumoniae, C. psittaci and C. trachomatis) have been characterized, and their structures are shown to resemble the rough form of enterobacterial LPS (such as Escherichia coli R mutants and Salmonella minnesota R595) (15–21). Of note, Chlamydiaceae LPS possesses a unique lipid A structure with longer (C18–C22) but fewer acyl chains (3–5) compared with a typical enterobacterial LPS such as E. coli with short hexaacyl chains (C12 and C14). In addition, Chlamydiaceae LPS has a weak endotoxic activity compared with E. coli LPS. Thus, the low biological activities of Chlamydiaceae LPS could permit the relatively weak host immune cell responses during Chlamydiaceae infection, which enables the persistent infection in the host cells.

However, it is still unclear why the biological activities of Chlamydiaceae LPS is low. Thus, to address this issue, we here investigated the actions of Chlamydiaceae LPS on PBMCs using C. psittaci LPS as an immunomodulator. The results have demonstrated that the LPS binds to monocytes and induces pro-inflammatory cytokine production from PBMC via the actions on LPS-binding protein (LBP) and the surface receptors CD14 and Toll-like receptor 4 (TLR4); however, C. psittaci LPS was much less potent than E. coli LPS in both the LPS binding and cytokine production. We further provided the evidence that C. psittaci LPS bound to both LBP and CD14 with lower affinities compared with E. coli LPS. Our findings suggest that the reduced affinities of Chlamydiaceae LPS for LPS-binding molecules may define the weak biological activities against host immune cells, thereby contributing to the chronic (persistent) inflammatory reactions during Chlamydiaceae infection.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
LPS and reagents
Chlamydophila LPS was prepared as described by Hussein et al. (16) from elementary bodies (EBs) of C. psittaci 6BC and provided by Professor Rudolf Toman (Slovak Academy of Science, Slovak Republic). Briefly, C. psittaci EBs, which were propagated in yolk sacs of embryonated hen eggs, were suspended in 2 M NaCl (20% v/v), inactivated with 0.2% formaldehyde and centrifuged at 20 000 x g. The pellet was suspended in PBS, digested with trypsin and then extracted with diethyl ether. EBs in the aqueous phase were precipitated by centrifugation, and LPS was extracted from EBs by a phenol–water method and further purified by a preparative PAGE (22). LPS from E. coli O111:B4 was obtained from Sigma-Aldrich (St Louis, MO). Alexa Fluor 488 (Alexa488)-labeled E. coli O55:B5 was obtained from Invitrogen (Carlsbad, CA, USA). Biotinylation of C. psittaci and E. coli LPS was performed using biotin-LC-hydrazide (Pierce, Rockford, IL, USA) according to manufacturer's protocol. For quantification, biotinylated LPS was separated by 10% SDS-PAGE, stained with a Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit (Invitrogen) and quantitated by comparison with the defined amounts of unlabeled LPS. The molecular weight of C. psittaci LPS (rough type) was estimated as 4500 by SDS-PAGE, whereas the smooth-type LPS from E. coli O111:B4 is estimated as the average molecular weight of 15 800 (23).

Neutralizing murine anti-human mAbs used were as follows: anti-CD14 (clone MY4; Beckman-Coulter, Fullerton, CA, USA), anti-TLR4 (clone HTA125; MBL, Nagoya, Japan), anti-CD11b (clone 44; Sigma-Aldrich) and anti-CD11b (clone D12; BD Biosciences, San Jose, CA, USA). Murine FITC-labeled anti-human CD3 (clone UCHT1) and PE-labeled anti-CD19 (clone HIB19) mAbs were purchased from BD Biosciences. Murine anti-human LBP mAb (clone 6G3) was obtained from HyCult Biotechnologies (Uden, Netherlands). Murine control antibodies (IgG) were obtained from eBioscience (San Diego, CA, USA) and Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Streptavidin–PE conjugate was from eBioscience, and streptavidin–HRP conjugate was from Zymed Laboratories (South San Francisco, CA, USA). Recombinant human LBP, CD14 and TLR4–MD-2 were purchased from R&D Systems (Minneapolis, MN, USA).

Preparation of PBMC
Human PBMCs from healthy donors were obtained by density-gradient centrifugation with Lymphoprep (Axis-Shield, Dundee, UK). The purity of PBMC (>95%) was confirmed by FACS staining for CD3, CD19 and CD14, in addition to the microscopic examination of Diff-Quick-stained cells. PBMCs were suspended in AIM-V serum-free medium (Invitrogen) containing 100 U ml–1 penicillin and 0.1 mg ml–1 streptomycin (Sigma-Aldrich). In some experiments, FCS (<0.03 ng ml–1 endotoxin; Equitech-Bio, Kerrville, TX, USA) was added to the culture medium.

Binding of LPS to PBMC
PBMCs (1 x 106 cells ml–1) were incubated with biotinylated C. psittaci LPS (1000 ng ml–1) or E. coli LPS (100 ng ml–1) in the presence or absence of LBP (10 and 100 ng ml–1) or 1% FCS at 37°C for 15 min with gentle shaking. Subsequently, cells were washed with PBS and incubated with streptavidin–PE for 30 min on ice. After washing twice with PBS, cells were analyzed with a FACScan and CellQuest Pro Software (BD Biosciences). For blocking experiments, PBMCs were pre-incubated with indicated concentrations of neutralizing mAbs (4 µg ml–1 anti-CD14, 10 µg ml–1 anti-TLR4 or 2.5 µg ml–1 anti-CD11b) or 10 µg ml–1 control IgG at 37°C for 15 min. Alternatively, PBMCs were incubated with excess amounts of unlabeled C. psittaci LPS or E. coli LPS (100 µg ml–1) in the presence of 10 ng ml–1 LBP at 37°C for 15 min. Then, biotinylated C. psittaci LPS (1000 ng ml–1) or Alexa488-labeled E. coli LPS (100 ng ml–1) was added and incubated for another 15 min. In the case of biotinylated C. psittaci LPS, cells were washed and further stained with streptavidin–PE.

Cytokine production from LPS-stimulated PBMC
To measure the production of IL-6, IL-1β and TNF-{alpha} from LPS-stimulated PBMC, cells (1 x 106 cells ml–1 in 96-well plates) were treated with or without neutralizing mAbs (anti-CD14, anti-TLR4 or anti-CD11b; 2.5–10 µg ml–1) or control IgG (10 µg ml–1) in AIM-V medium containing 1% FCS at 37°C for 1 h and then stimulated with C. psittaci LPS (100 and 1000 ng ml–1) or E. coli LPS (1, 10 and 100 ng ml–1) for 6 h. Culture supernatants were collected and stored at –80°C for further assay. The amounts of IL-6, IL-1β and TNF-{alpha} in the culture supernatants were determined using Ready-To-Go Cytokine ELISA kit (eBioscience). The detection limits of IL-6, IL-1β and TNF-{alpha} were 15.6 pg ml–1.

Interaction of LPS with LBP
The binding of biotinylated LPS to immobilized LBP was measured as described by Scott et al. (24). Anti-human LBP mAb (6G3), which recognizes LBP molecules as well as LBP–LPS complexes, was used as a capture mAb. The mAb was diluted to 1 µg ml–1 in 0.1 M NaHCO3 (pH 9.0) and absorbed to 96-well ELISA plates (Corning, Acton, MA, USA) overnight at 4°C. The plates were blocked with PBS containing 1% BSA for 2 h at room temperature. After washing with PBS–0.05% Tween 20, LBP in PBS containing 0.1% BSA was added to the plates at indicated concentrations (0.1–200 ng ml–1) for 1.5 h at room temperature. Then, the plates were washed, and biotinylated C. psittaci LPS (1000 ng ml–1, 220 nM) or E. coli LPS (100 ng ml–1, 6.3 nM) diluted in PBS containing 0.1% BSA was incubated in the plates for 1 h at room temperature. The LPS bound to immobilized LBP was detected using streptavidin–HRP (1:2500 dilution; Zymed Laboratories) and tetramethylbenzidine (TMB) substrate (Sigma-Aldrich). For competition experiments, unlabeled C. psittaci LPS (0.22 nM–22 µM) or E. coli LPS (0.063 nM–6.3 µM) was pre-incubated in the LBP (100 ng ml–1)-captured plates for 30 min, and then biotinylated C. psittaci LPS or E. coli LPS was added.

Interaction of LPS with CD14
The interaction of LPS with CD14 was analyzed by the binding of biotinylated LPS to immobilized CD14 (25). Briefly, ELISA plates were coated with 2.5 µg ml–1 CD14 in 0.1 M NaHCO3 (pH 9.0) overnight at 4°C. The plates were blocked with 1x Block Ace (Dainippon Pharmaceutical, Tokyo, Japan) for 2 h at room temperature. After washing with PBS–0.05% Tween 20, biotinylated C. psittaci LPS (1000 ng ml–1, 220 nM) or E. coli LPS (100 ng ml–1, 6.3 nM) diluted in PBS containing 0.5% BSA was incubated in the plates in the presence or absence of LBP for 1 h at 37°C. Subsequently, the LPS bound to CD14 was detected using streptavidin–HRP and TMB substrate. For competition assay, anti-CD14 mAb (MY4; 5 µg per well), polymyxin B (PMB) sulfate (5 µg per well; Sigma-Aldrich) or unlabeled C. psittaci LPS (0.22 nM–22 µM) or E. coli LPS (0.063 nM–6.3 µM) was pre-incubated with 100 ng ml–1 LBP in the CD14-immobilized plates for 30 min, and then biotinylated C. psittaci LPS or E. coli LPS was added.

Interaction of LPS with TLR4–MD-2
ELISA plates were coated with recombinant TLR4–MD-2 (2 µg ml–1) in PBS at 4°C overnight. After blocking with PBS containing 2% BSA for 1 h, the plates were washed with PBS–0.05% Tween 20. Biotinylated C. psittaci LPS or E. coli LPS (10–1000 ng ml–1) diluted in PBS–0.5% BSA was incubated in the plates in the presence or absence of 100 ng ml–1 LBP for 1 h at 37°C. Then the binding of LPS to TLR4–MD-2 was determined using streptavidin–HRP and TMB substrate. Inhibition of the binding of biotinylated LPS to TLR4–MD-2 was assessed by the addition of unlabeled LPS (C. psittaci: 0.22 nM–22 µM; E. coli: 0.063 nM–6.3 µM) as described above.

Statistical analysis
The data were expressed as the mean ± SD and analyzed for significant difference by a one-way analysis of variance and a post-hoc test using the StatView program (SAS, Berkeley, CA, USA). Differences were considered statistically significant if a P-value < 0.05. For competition analysis, curve fitting and 50% inhibitory concentration (IC50) determination was performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
LBP-dependent binding of C. psittaci LPS to human peripheral blood monocytes
LBP facilitates the binding of LPS to mononuclear phagocytes (monocytes/macrophages) by catalyzing transfer of LPS monomers to CD14 molecules (26, 27). Thus, we first evaluated the binding ability of C. psittaci LPS to human peripheral blood monocytes in the presence or absence of LBP using flow cytometry. As shown in Fig. 1(A), in the absence of LBP, biotinylated C. psittaci LPS (1000 ng ml–1) somewhat bound to monocytes, and the addition of LBP (10 ng ml–1) increased the binding. Similarly, the binding of biotinylated E. coli LPS (100 ng ml–1) to monocytes was enhanced in the presence of LBP (Fig. 1D); however, the binding level of C. psittaci LPS was much lower than that of E. coli LPS. Almost the same results were observed by the addition of 1% FCS (data not shown). These results indicated that C. psittaci LPS could bind to monocytes in an LBP-dependent manner, although the binding of C. psittaci LPS was apparently less than that of E. coli LPS.


Figure 1
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  		Fig. 1. Flow cytometric analysis of the binding of Chlamydophila psittaci LPS to human monocytes. PBMCs (1 x 106 cells ml–1) suspended in AIM-V serum-free medium were incubated with biotin-labeled C. psittaci LPS (Biotin-cLPS, 1000 ng ml–1) or Escherichia coli LPS (Biotin-eLPS, 100 ng ml–1) in the presence or absence of LBP (10 ng ml–1) (A and D). PBMCs were also incubated with Biotin-cLPS (1000 ng ml–1) in the presence or absence of excess amounts of unlabeled C. psittaci LPS (cLPS; 100 µg ml–1, 100-fold molar excess) or E. coli LPS (eLPS; 100 µg ml–1; 28.6-fold molar excess) (B and C). Thereafter, the cells were incubated with streptavidin–PE (SA-PE). Alternatively, PBMCs were incubated with Alexa488-labeled E. coli LPS (Alexa-eLPS, 100 ng ml–1) in the presence of 10 ng ml–1 LBP with or without excess amounts of unlabeled eLPS (10 µg ml–1; 100-fold molar excess) or cLPS (10 µg ml–1; 349-fold molar excess) (E and F). For each samples, 10 000 monocytes were analyzed by flow cytometry. BG, background fluorescence of unlabeled cells; SA-PE, fluorescence of cells stained with SA-PE only.

 
Next, to characterize the LPS receptors on monocytes, we performed a competition assay. The addition of both unlabeled C. psittaci LPS (100-fold molar excess) and E. coli LPS (28-fold molar excess) blocked the binding of biotinylated C. psittaci LPS to monocytes (>48 and >50% inhibition, respectively) (Fig. 1B and C). Similarly, unlabeled C. psittaci LPS (355-fold molar excess) partially blocked the binding of Alexa488-labeled E. coli LPS to monocytes (62.7% inhibition); however, unlabeled E. coli LPS (100-fold molar excess) markedly inhibited the binding of labeled E. coli LPS (97.5% inhibition) (Fig. 1E and F). These findings suggest that C. psittaci LPS shares the common receptors with E. coli LPS on monocytes, but that the binding affinity for C. psittaci LPS may be lower than that for E. coli LPS.

It has been reported that several cellular receptors such as TLR4 and CD11b as well as CD14 form the clusters on cells and are involved in the LPS signaling (28–31). Thus, we investigated the surface receptors for C. psittaci LPS on monocytes using neutralizing mAbs. Anti-CD14 and anti-TLR4 mAbs significantly inhibited the binding of C. psittaci LPS to monocytes by 41.8 and 47.4%, respectively (Fig. 2A). Anti-CD11b mAb also inhibited the C. psittaci LPS binding by 61%. In contrast, the binding of E. coli LPS to monocytes was significantly blocked by anti-CD14 and anti-TLR4 mAbs (95 and 27.4%, respectively), but not by anti-CD11b mAbs (Fig. 2B).


Figure 2
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  		Fig. 2. Effect of blocking mAbs on the binding of Chlamydophila psittaci LPS to monocytes. PBMCs (1 x 106 cells ml–1) suspended in AIM-V containing 10 ng ml–1 LBP were pre-incubated without (control) or with blocking mAbs (2.5–10 µg ml–1) or control IgG (10 µg ml–1) for 15 min and then added with biotinylated C. psittaci LPS (1000 ng ml–1, A) or Escherichia coli LPS (100 ng ml–1, B). The mean fluorescent intensity of bound LPS was analyzed by flow cytometry. LPS binding was expressed as a percentage of control. Data are the mean ± SD of three independent experiments. Values are compared between the incubation in the absence (control) and presence of blocking mAbs or control IgG. *P < 0.05, **P < 0.005.

 
Pro-inflammatory cytokine production from C. psittaci LPS-stimulated PBMC
It is known that enterobacterial LPS from E. coli and Salmonella elicits the production of pro-inflammatory cytokines such as IL-1β, TNF-{alpha} and IL-6 from PBMC via the actions on CD14 and TLR4 (13). Thus, we investigated whether C. psittaci LPS could also activate PBMC to produce pro-inflammatory cytokines.

Following the stimulation with C. psittaci LPS (100 and 1000 ng ml–1), IL-6 was produced from PBMC in a dose-dependent manner, and the production of these cytokines was significantly enhanced by the addition of LBP (Fig. 3A). However, in agreement with the LPS binding (Fig. 1), the cytokine production by C. psittaci LPS was much lower than that by E. coli LPS (1 and 10 ng ml–1, Fig. 3B). Moreover, experiments using neutralizing antibodies demonstrated that C. psittaci LPS-induced production of IL-6 was blocked by not only anti-CD14 mAb but also by anti-TLR4 mAb (Fig. 3C). Interestingly, anti-CD11b mAb, which inhibited the binding of C. psittaci LPS, had no effect on the cytokine production. In contrast, anti-CD14 and anti-TLR4 mAbs inhibited E. coli LPS-induced cytokine production as well as LPS binding (Fig. 3D). Similar results were observed for IL-1β and TNF-{alpha} production (data not shown).


Figure 3
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  		Fig. 3. IL-6 production from Chlamydophila psittaci LPS-stimulated PBMC. PBMCs (1 x 106 cells ml–1) suspended in AIM-V were stimulated with C. psittaci LPS (cLPS; 100 and 1000 ng ml–1; A) or Escherichia coli LPS (eLPS; 1 and 10 ng ml–1; B) in the absence (–) or presence of LBP (10 and 100 ng ml–1) for 6 h. For neutralization experiments, PBMCs suspended in AIM-V containing 1% FCS were incubated without (control) or with appropriate mAbs (2.5–10 µg ml–1) or control IgG (10 µg ml–1) for 1 h prior to LPS stimulation (C and D). Amounts of IL-6 in culture supernatants were measured by ELISA. Alternatively, PBMCs were incubated without cLPS or eLPS in the presence or absence of LBP (Rest). Data are the mean ± SD of five to seven independent experiments. Values are compared between the incubation without (–) and with LBP (A and B) or the absence (control) and presence of blocking mAbs or control IgG (C and D). ND, not detected. *P < 0.05, **P < 0.005.

 
These observations suggest that CD14 and TLR4 but not CD11b are involved in the pro-inflammatory cytokine production from C. psittaci LPS-stimulated PBMC.

Interaction of C. psittaci LPS with LBP and CD14
The above observations clearly indicate that the biological activities of C. psittaci LPS are lower than those of E. coli LPS. To elucidate these differences, we focused on the interaction of C. psittaci LPS with the two major LPS-binding molecules LBP and CD14.

As shown in Fig. 1, LBP enhanced the binding of C. psittaci LPS to monocytes, suggesting the interaction of C. psittaci LPS with LBP. Consistent with this, biotinylated C. psittaci LPS (1000 ng ml–1) directly bound to immobilized LBP (≥50 ng ml–1 LBP), and the binding reached a plateau at ≥100 ng ml–1 LBP (Fig. 4A). In contrast, biotinylated E. coli LPS (100 ng ml–1) bound to immobilized LBP (≥10 ng ml–1), and the binding reached a plateau at ≥50 ng ml–1 LBP. Apparently, the binding levels of E. coli LPS were higher than those of C. psittaci LPS. We subsequently performed the competitive ELISA to determine the binding affinities of C. psittaci LPS and E. coli LPS to immobilized LBP (100 ng ml–1). Both unlabeled C. psittaci LPS and E. coli LPS effectively inhibited the binding of biotinylated C. psittaci LPS (1000 ng ml–1, i.e. 220 nM) to LBP with IC50 values of 8.9 and 0.4 nM, respectively (Fig. 4B). Similarly, unlabeled C. psittaci LPS and E. coli LPS dose dependently inhibited the binding of biotinylated E. coli LPS (100 ng ml–1, i.e. 6.3 nM) to immobilized LBP (IC50 values of 230.5 and 43.2 nM, respectively, Fig. 4C). Of note, C. psittaci LPS was approximately 5- to 20-fold less potent than E. coli LPS in suppressing the binding of biotinylated LPS (C. psittaci and E. coli) to LBP.


Figure 4
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  		Fig. 4. Binding profiles of Chlamydophila psittaci LPS to immobilized LBP. (A) Increasing amounts of LBP (0.1–200 ng ml–1) were immobilized to anti-LBP mAb-coated microplates, and biotinylated C. psittaci LPS (Biotin-cLPS; 1000 ng ml–1, 220 nM) or Escherichia coli LPS (Biotin-eLPS; 100 ng ml–1, 6.3 nM) was added to the plates. (B) Biotin-cLPS (1000 ng ml–1, 220 nM) was incubated in the LBP (100 ng ml–1, 6.3 nM)-immobilized plates in the absence or presence of unlabeled C. psittaci LPS (0.22 nM–22 µM) or E. coli LPS (0.063 nM–6.3 µM). (C) Biotin-eLPS (100 ng ml–1, 6.3 nM) was incubated in the LBP (100 ng ml–1, 6.3 nM)-immobilized plates in the absence or presence of unlabeled C. psittaci LPS (0.22 nM–2.2 µM) or E. coli LPS (0.063–630 nM). Finally, bound LPS was detected by the sequential addition of streptavidin–HRP, TMB and 1 M H2SO4. Data are the mean ± SD of three independent experiments.

 
We further analyzed the interaction of C. psittaci LPS with CD14. The binding of biotinylated C. psittaci LPS (1000 ng ml–1) to immobilized CD14 was enhanced in the presence of increasing concentrations of LBP (Fig. 5A). Of importance, neutralizing anti-CD14 mAb almost completely blocked the binding of C. psittaci LPS to immobilized CD14 in the presence of LBP (Fig. 5B). Furthermore, PMB, a cationic cyclic peptide, which binds to lipid A moiety and prevents LPS–LBP interaction, also abolished the C. psittaci LPS binding to CD14. These findings clearly indicate that C. psittaci LPS can bind to CD14 in an LBP-dependent manner. Similarly, the binding of biotinylated E. coli LPS (100 ng ml–1) to immobilized CD14 was enhanced by increasing concentrations of LBP, and the binding was significantly inhibited by anti-CD14 mAb and PMB (Fig. 5A and B). Next, we assessed the binding affinity of C. psittaci LPS to CD14 in the presence of LBP. As shown in Fig. 5(C), unlabeled C. psittaci LPS and E. coli LPS inhibited the binding of biotinylated C. psittaci LPS (220 nM) to CD14 in a dose-dependent fashion (IC50 values of 335.0 and 12.8 nM, respectively). Likewise, unlabeled C. psittaci LPS and E. coli LPS blocked the binding of biotinylated E. coli LPS (6.3 nM) to CD14 with IC50 values of 81.3 and 12.3 nM, respectively (Fig. 5D).


Figure 5
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  		Fig. 5. Binding profiles of Chlamydophila psittaci LPS to immobilized CD14. (A) CD14 (2.5 µg ml–1) was absorbed to the microplates. After blocking, biotinylated C. psittaci LPS (Biotin-cLPS, 1000 ng ml–1) or Escherichia coli LPS (Biotin-eLPS, 100 ng ml–1) was incubated in the presence of the indicated concentration of LBP. (B) The CD14-immobilized plates were incubated without (control) or with neutralizing anti-CD14 (5 µg per well) or PMB (5 µg ml–1) in the presence of 100 ng ml–1 LBP and Biotin-cLPS (1000 ng ml–1) or E. coli LPS (100 ng ml–1). (C) Biotin-cLPS (1000 ng ml–1, 220 nM) was incubated with 100 ng ml–1 LBP in the CD14-immobilized plates in the absence or presence of unlabeled C. psittaci LPS (0.22 nM–22 µM) or E. coli LPS (0.063 nM–6.3 µM). (D) Biotin-eLPS (100 ng ml–1, 6.3 nM) was incubated with 100 ng ml–1 LBP in the CD14-immobilized plates in the absence or presence of unlabeled C. psittaci LPS (0.22 nM–2.2 µM) or E. coli LPS (0.063–630 nM). Bound LPS was finally detected using streptavidin–HRP. Data are the mean ± SD of three independent experiments. In panel B, values are compared between the absence (control) and presence of anti-CD14 mAb or PMB. *P < 0.05, **P < 0.005.

 
Taken together, these observations indicate that the binding affinities of C. psittaci LPS for both LBP and CD14 are much lower than those of E. coli LPS.

Interaction of C. psittaci LPS with TLR4–MD-2 complex
It is generally accepted that LPS captured by CD14 is transferred to TLR4–MD-2 signaling complex (28, 30). Thus, we finally investigated the binding of C. psittaci LPS to immobilized TLR4–MD-2 complex using ELISA and compared with that of E. coli LPS.

As shown in Fig. 6(A), both C. psittaci LPS and E. coli LPS bound to immobilized TLR4–MD-2 in an LBP-dependent manner. However, the binding of C. psittaci LPS was much lower than that of E. coli LPS, which can be explained by the lower affinity of C. psittaci LPS for LBP as compared with E. coli LPS (Fig. 4A).


Figure 6
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  		Fig. 6. Binding of Chlamydophila psittaci LPS to immobilized TLR4–MD-2 complex. (A) TLR4–MD-2 complex (2 µg ml–1) was absorbed to the microplates. After blocking, biotinylated C. psittaci LPS (Biotin-cLPS) or Escherichia coli LPS (Biotin-eLPS) was incubated in the absence (–LBP) or presence (+LBP) of 100 ng ml–1 LBP. (B) Biotin-cLPS (1000 ng ml–1, 220 nM) was incubated with 100 ng ml–1 LBP in the TLR4–MD-2-immobilized plates in the absence or presence of unlabeled C. psittaci LPS (0.22 nM–22 µM) or E. coli LPS (0.063 nM–6.3 µM). (C) Biotin-eLPS (100 ng ml–1, 6.3 nM) was incubated with 100 ng ml–1 LBP in the TLR4–MD-2-immobilized plates in the absence or presence of unlabeled C. psittaci LPS (0.22 nM–2.2 µM) or E. coli LPS (0.063–630 nM). Bound LPS was finally detected using streptavidin–HRP. Data are the mean ± SD of five independent experiments.

 
Further, the competition assay indicated that unlabeled LPS dose dependently inhibited the binding of biotinylated C. psittaci or E. coli LPS to TLR4–MD-2 complex (Fig. 6B and C). However, in contrast with the cases of LBP and CD14 (Figs 4B and C and 5B and C), unlabeled C. psittaci LPS and E. coli LPS blocked the binding of biotinylated C. psittaci LPS to TLR4–MD-2 (Fig. 6B, IC50 values of 1.40 and 1.81 nM, respectively) and the binding of biotinylated E. coli LPS to TLR4–MD-2 (Fig. 6C, IC50 values of 7.20 and 4.14 nM, respectively) with similar affinities.

These observations suggest that the weak biological activity of C. psittaci LPS can be defined by the low capacity of C. psittaci LPS to bind with LBP and CD14 rather than TLR4–MD-2 complex.


   Discussion
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 Abstract
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 Methods
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 Discussion
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 References
 
Chlamydiaceae can survive in the infected cells, and persistently activate host cells at the infection site, thereby inducing sustained immunological response (3, 7, 13, 32–34). To date, Chlamydiaceae-derived factors such as LPS, MOMP and HSP60 are reported to be potential activators of host cells (7–10).

In this report, we first demonstrated that C. psittaci LPS, a Chlamydiaceae LPS, can directly bind to human monocytes in an LBP-dependent manner (Fig. 1), although its structure is distinct from enterobacterial LPS (16, 17, 19). Furthermore, competition assays suggested that C. psittaci LPS shares the common surface receptors with E. coli LPS on monocytes; excess E. coli LPS almost completely inhibited the binding of C. psittaci LPS to monocytes and vice versa. Moreover, neutralizing mAbs against LPS receptors (CD14 and TLR4) significantly blocked the binding of C. psittaci LPS as well as E. coli LPS (Fig. 2). These results clearly indicate that C. psittaci LPS can interact with CD14 and TLR4, despite its unique structure. Consistent with our observation, Heine et al. also demonstrated that LPS from C. trachomatis E could activate HEK293 cells expressing CD14 and TLR4–MD-2, suggesting that Chlamydiaceae-derived LPS can recognize CD14 and TLR4 as with enterobacterial LPS. We further provided the evidence that C. psittaci LPS is capable of not only binding to PBMC but also activating them to produce cytokines, although C. psittaci LPS is less potent than E. coli. Moreover, consistent with the LPS binding, the production of pro-inflammatory cytokines (IL-6, IL-1β and TNF-{alpha}) from PBMC was augmented by LBP (Fig. 3A and C and data not shown) and significantly inhibited by anti-CD14 and anti-TLR4 (Fig. 3B and D).

Previous studies showed that certain receptors facilitate the LPS binding to CD14 and TLR4 (14,28–30). In this context, CD11b, an {alpha} subunit of β2 integrin complement receptor type 3 (also called Mac-1) is associated with CD14 and TLR4 (35–38). Interestingly, neutralizing anti-CD11b mAb substantially inhibited the binding of C. psittaci LPS to monocytes, suggesting that CD11b is involved in the LPS binding (Fig. 2A). However, anti-CD11b, which blocked the binding of C. psittaci LPS to monocytes, did not interfere with the cytokine production. In agreement with our findings, it has been revealed that extracellular domain of Mac-1 is involved in the LPS binding but cytoplasmic domain is not required for LPS-mediated intracellular signaling (36). Thus, CD11b may only facilitate the binding of C. psittaci LPS to the cell surface.

Recent studies have suggested that Chlamydiaceae-induced production of IFN-{gamma} is regarded as one of the potential factors modulating the pathogen growth and regulated by IL-18, an IFN-{gamma}-inducing factor (33, 34, 39–41). In this regards, we have evaluated the production of these cytokines from C. psittaci LPS-stimulated PBMC. However, negligible amounts of IL-18 and IFN-{gamma} could be produced from C. psittaci LPS-stimulated PBMC (data not shown). Thus, the IFN-{gamma} and IL-18 production is likely to be mainly regulated by other Chlamydophila-derived constituents than LPS. Identification of those constituents should be important for understanding of pathogen growth during Chlamydiaceae infection.

It is noteworthy that the binding capacities and biological activities of C. psittaci LPS were lower than those of E. coli LPS (Figs 1–3GoGo). Thus, we postulated that the lower activities of C. psittaci LPS may result from its weak interaction with LBP and/or receptor molecules. By employing the ELISA-based assays, we confirmed not only the direct binding of C. psittaci LPS to LBP but also the LBP-dependent interaction of LPS with CD14, which is in good agreement with the LPS binding to monocytes analyzed by flow cytometry (Figs 1, 4 and 5Go). Supporting our hypothesis, the binding of C. psittaci LPS to LBP and CD14 was much less than that of E. coli LPS. Indeed, higher concentrations of C. psittaci LPS were required for inhibiting the binding of E. coli to LBP and CD14, while E. coli LPS effectively competed with the binding of C. psittaci at the low concentrations (Figs 4 and 5). Based on IC50 values, the binding affinity of C. psittaci LPS for LBP was at least 5-fold lower than that of E. coli LPS, and the binding affinity for CD14 is at least 6-fold lower than E. coli LPS. Thus, it is reasonable to speculate that the structural differences between C. psittaci and E. coli LPS may affect the binding profiles of these LPS. Furthermore, the low binding affinities of C. psittaci LPS with LBP and CD14 are assumed to reduce the efficiency of LPS-induced signaling.

Moreover, the ELISA-based binding experiments revealed that C. psittaci LPS and E. coli LPS can directly bind to TLR4–MD-2 complex in an LBP-dependent manner, and the binding of C. psittaci LPS was much lower than that of E. coli LPS. Interestingly, however, the competition experiments showed that both unlabeled C. psittaci LPS and E. coli LPS inhibited the binding of biotinylated C. psittaci LPS and E. coli LPS to TLR4–MD-2 complex with similar affinities. Taken together, the low binding affinities of C. psittaci LPS with LBP and cell-surface CD14 but not TLR4–MD-2 are most likely to define its weak bioactivities.

It has been proposed that persistent infection with Chlamydiaceae in host cells is closely associated with chronic inflammation (3, 7, 33, 34). In the present study, we have suggested that C. psittaci LPS play a role in inflammatory responses via the weak activation of host immune cells such as PBMC. Recent study indicated that Chlamydiaceae-derived unknown components can be recognized by nucleotide-binding oligomerization domain 1 (NOD1) to activate endothelial cells and induce IL-18 production (42, 43). Thus, Chlamydiaceae LPS and other components are likely to cooperatively stimulate host cells and contribute to the persistent infection. It still remains unknown whether Chlamydiaceae LPS can activate other target cells such as endothelial cells, which differently express LPS receptors from monocytes/macrophages. Detailed analysis of the actions of Chlamydiaceae LPS on these cells leads to a better understanding of Chlamydiaceae-induced immune responses in the host.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
Institute for Environmental and Gender-specific Medicine, Juntendo University School of Medicine; High Technology Research Center, Juntendo University; Scientific Research from Japan Society for the Promotion of Science; 21st Century COE Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.


   Abbreviations
 
Alexa488, Alexa Fluor 488
EB, elementary body
HSP60, heat shock protein 60
IC50, 50% inhibitory concentration
LBP, LPS-binding protein
MOMP, major outer membrane protein
PMB, polymyxin B
TLR4, Toll-like receptor 4
TMB, tetramethylbenzidine

   Notes
 
Transmitting editor: K. Okumura

Received 26 December 2006, accepted 8 November 2007.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

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