Chlamydophila pneumoniae induces expression of Toll-like Receptor 4 and release of TNF-α and MIP-2 via an NF-κB pathway in rat type II pneumocytes

C. pneumoniae strain TW183 was cultured and purified as described previously [13]. Briefly TW183 was grown to high titers in cycloheximide-treated HEp-2 cells. Infected monolayers were harvested from culture flasks and sonicated for 30 sec. Cellular debris was removed by centrifugation at 500 × g for 10 min at 4°C. Titer determination in cycloheximide-treated HEp-2 cells was performed with a thawed aliquot (in triplicate). Aliquots diluted with an equal volume of sucrose-phosphate-glutamate buffer supplemented with 10% foetal calf serum (FCS) were stored at -80°C until use. C. pneumoniae concentrations used were expressed as inclusion forming units (IFU)/ml. Type II pneumocytes were isolated from the lungs of adult male Wistar rats (body weight 120–140 g) according to Dobbs et al. [14].

Rat alveolar macrophages were separated from bronchial lavage fluid. The lungs were lavaged six times with phosphate-buffered saline (PBS) containing 0.2 mM EGTA (ethylene glycol tetraacetic acid). Cells were then collected by centrifugation at 230 × g for 10 min. The cell pellet was re-suspended in modified Eagle medium (MEM) (PAA laboratories GmbH, Linz, Austria).

Isolated type II cells were pre-incubated with the following substances: mevastatin (10 μM), also known as compactin, a specific inhibitor of the Rho-GTPase family; the calcium chelator BAPTA/AM (5 μM); SN50 (50 μg/ml), a cell-permeable peptide that inhibits the nuclear translocation of NF-κB/Rel complexes; SN50M (50 μg/ml), a cell-permeable control peptide; parthenolide (25 μM), an inhibitor of NF-κB through prevention of IκB phosphorylation (all chemicals were obtained from Calbiochem, Darmstadt, Germany) or MEM with corresponding concentrations of dimethyl sulfoxide (DMSO). Bacteria were treated with polymyxin obtained from Sigma, Deisenhofen, Germany. Thereafter, type II cells and macrophages in MEM in absence of FCS were incubated with or without C. pneumoniae with a multiplicity of infection (MOI) of 2 (2 × 10⁶ IFU per 1 × 10⁶ target cells) as used in previous experiments [12].

The viability of isolated alveolar macrophages or type II cells was assessed by trypan blue staining and was more than 95% or 98% respectively and remained above 94% after cell incubation with or without C. pneumoniae and different inhibitors.

Cell purity, estimated by hematoxylin staining, was more than 95% or 92% respectively.

Cellular RNA was extracted from 5 × 10⁵ type II cells using an RNeasy™ extraction kit (Quiagen, Hilden, Germany) according to the manufacturers instructions. RNA concentration was quantified by determining optical density at 260 and 280 nm (Ultraspec 2000 Spectrophotometer, Pharmacia Biotech, Freiburg, Germany) and adjusted to 100 pg/μl.

Semi-quantitative polymerase chain reaction was used to assess TLR2 and TLR4 mRNA expression under different conditions in rat alveolar type II cells. The mRNA expression levels of TLR2 and TLR4 genes were determined in total RNA from pooled samples of three independent experiments. Reverse transcription (RT) of 1 μg RNA into cDNA was performed by using random hexamers, AMV reverse transcriptase and 1× PCR buffer (20 mM Tris-HCL, pH 8.3, 50 mM KCl, 2 mM MgCl, 100 μg/ml bovine serum albumin). RT was performed at 42°C for 30 min followed by 95°C for 5 min to inactivate the reverse transcriptase enzyme. The cDNA was amplified by PCR in a total volume of 50 μl using 2.5 U Ampli-Taq DNA polymerase, 100 μM dATP, dCTP, dGTP, 50 μM dTTP and 0.5 μM of each primer in 1× PCR buffer. Incorporation of digoxigenin was performed by addition of 10 μM digoxigenin-11-dUTP to the PCR reaction mixture. For GAPDH housekeeping gene, PCR was performed with 25 cycles of 1 minute each at 95°C, 54°C and 72°C in a microprocessor-driven thermal cycler (Fa. Landgraf, Hannover, Germany). For TLR2 and TLR4 amplification, PCR was performed with 35 or 40 cycles of 1 minute each at 95°C, 65°C and 72°C.

Primer sequences for GAPDH were (GenBank BC059110; product size 303 bp):

Products were stained with ethidium bromide and electrophoresed through a 1.5% agarose gel. The PCR products were transferred to nylon membranes by capillary blotting using 20× SSC as blotting buffer. The nylon membranes were fixed by UV light and the digoxigenin-UTP that had been incorporated into the PCR products was visualized by staining with anti-digoxigenin antibody conjugated to alkaline phosphatase [15]. Luminescence of the substrate (Lumigen™ PPD) was documented by short exposure to X-ray film.

Densitometric calculations of digital film images were performed with the analysis program Scion Image (Scion Corporation, Frederick, MD).

Isolated type II cells were incubated with C. pneumoniae in suspension. Nuclear and cytoplasmatic extracts and membrane fractions were prepared from cells as reported previously [16, 17]. The protein content of nuclear, cytoplasmatic and membrane fraction extracts was determined by Bradford assay using bovine serum albumin (BSA) as a standard method (Bio-Rad Laboratories protein assay kit, Richmond, USA). Protein fractions were stored at -80°C. TLR4 protein was assayed in membrane and cytoplasmatic extracts, IκBα protein in cytoplasmatic extracts, NF-κB p65 protein in nuclear extracts and Rac1 and RhoA protein in membrane extracts of type II cells by Western blotting. Proteins (40 μg or 60 μg for TLR4, 40 μg for IκBα, 10 μg for p65 and 40 μg for Rac1 and RhoA) from each sample were mixed with 2 × sodium dodecyl sulfate (SDS) sample buffer, heated at 95°C for 5 minutes, and separated by 10% (TLR4, p65), 12.5% (IκBα) and 15% (Rac1 and RhoA) SDS-polyacrylamide gels. The separated proteins were blotted onto nitrocellulose membranes. Non-specific binding sites were blocked with 10% nonfat dry milk in Tris-buffer saline (TBS)-0.05% Tween. Goat IgG anti-TLR4 (L-14) sc-16240, mouse monoclonal IgG anti-IκBα (H-4) sc-1643, rabbit IgG anti-NF-κB p65 (C-20) sc-372, goat IgG anti-Rac1 (T-17) sc-6084 and rabbit IgG anti-RhoA (119) sc-179 antibodies (from Santa Cruz Biotechnology, Heidelberg, Germany) were used for primary detection. Peroxidase-conjugated anti-goat IgG, anti-mouse IgG and anti-rabbit IgG antibodies (Dianova, Hamburg, Germany) were employed for secondary detection. The specific protein bands were visualized on film by enhanced chemiluminescence (ECL) (Amersham Biosciences, Inc, Piscataway, NJ) according to the manufacturers. The bands were quantified by scanning densitometry using a GS-710 Imaging Densitometer (Bio-Rad, Hercules, CA).

All other standard reagents and chemicals were of analytical grade and obtained from different suppliers.

The isolated type II cells were plated on glass cover slips for 3 hours at 37°C.

For TLR4-binding experiments, type II cells were incubated with and without C. pneumoniae for 10 min at 37°C, washed and then without permeabilization incubated with goat anti-TLR4 antibody for 60 min at 22°C.

Cells were pre-incubated with or without drugs or with corresponding concentrations of DMSO and incubated with C. pneumoniae as described by Wissel et al. [12, 18]. Cells were fixed in 1% paraformaldehyde/250 mM Hepes (pH 7.4) and permeabilized with 0.04% saponin. For staining of TLR4 cells were incubated with goat anti-TLR4 antibody for 20 hours at 4°C. In experiments designed to block NF-κB activity, type II cells were pre-incubated with 20 μg/ml anti-TLR4 monoclonal antibody (HTA125, preservative free) or isotype control antibody (QBIOGENE-ALEXIS GmbH, Grünberg, Germany) for 30 min at 37°C before incubation with C. pneumoniae for 60 min at 37°C. To stain the NF-κB subunits within the cells, the following antibodies were used: mAb anti-IκBα and mAb anti-NF-κB p65 subunit, recognizing predominantly p65 only when IκBα is not bound to p65 (Chemicon, Hofheim, Germany) and incubated for 20 hours at 4°C. For staining of Rac1 and Rho protein cells were incubated with goat anti-Rac1 and rabbit anti-RhoA antibody for 2 hours at 22°C. After washing steps in PBS, cells were incubated with one of the following secondary antibodies: Alexa^® 488 or Alexa^® 594 anti-goat IgG, anti-mouse IgG labeled with Alexa^® 488 or Alexa^® 594, or with anti-rabbit IgG labeled with Alexa^® 594 (Molecular Probes Europe BV, Leiden, Netherlands) for 2 hours at 22°C. To study the F-actin cytoskeleton, Alexa^® 488-conjugated phalloidin (Molecular Probes) was used (2 hours at 22°C). Nuclear DNA was stained with 4',6-diamidino-2-phenylindole (DAPI) (Molecular Probes) for 20 minutes.

For TLR4-binding experiments samples were analyzed with a Leica CLSM (Leica Lasertechnik GmbH, Heidelberg, Germany), equipped with a krypton-argon laser as described previously [18]. Images were captured with a NPL Fluotar 40×/1.3 oil immersion objective. To visualize the cell boundaries, images were recorded simultaneously with bright-field contrast in relation with the channel system of CLSM in the equatorial plane of cells.

The triple staining was analyzed with a Zeiss laser scanning microscope LSM 510 META with Axiovert 200 M (Carl Zeiss Jena GmbH, Jena, Germany) as described previously [12]. Images were taken using a Plan-Apochromat 63×/1.4 oil immersion objective and fluorescence excitation at 488 nm (30 mW Ar-Laser), 543 nm (1 mW HeNe-Laser) and 405 nm (Diode Laser). To minimize signal crosstalk, a sequential scan with fast change of excitation lines was performed.

The semi-quantitative estimation of NF-κB subunits was made according to the methods described previously by Wissel et al. [12] and analyzed with Zeiss LSM 510 system with Axiovert microscope as described above. Thirty randomly chosen cells from six different regions were analyzed. Confocal settings were optimized and maintained for all images. Images were recorded with a scanning speed at 8s/frame with a resolution of 512 × 512 pixels. Serial sections of cells (z stack) at a depth of 0.5 μm were performed. Fluorescence intensity was determined on confocal images using a computer-based image analysis system equipped with MetaView™ software (obtained from Visitron Systems GmbH, Puchheim, Germany) by measuring the average intensity per pixel within a fixed-area circle placed over the relevant area of the cell.

Three-hour adherent type II cells and macrophages (2 × 10⁶ cells/ml) on plastic dishes were incubated with or without C. pneumoniae for different periods of time. Adherent type II cells were pre-incubated with BAPTA/AM, SN50, SN50M or parthenolide followed by incubated with C. pneumoniae for 3 to 6 hours. Cell culture supernatants were collected and analyzed for TNF-α and MIP-2 secretion by enzyme-linked immunosorbent assay (ELISA). The levels of TNF-α production were determined using rat TNF-α Quanticine ELISA (R&D Systems Inc, Minneapolis, USA). MIP-2 concentration in the samples was determined using rat MIP-2 ELISA kit (Biosource Europe, Nivelles, Belgium) following the manufacturer' s instructions.

Statistical comparisons were performed by analysis of variance (ANOVA) with subsequent Fisher's protected least significant difference (PLSD) test. The level of significance was set at p < 0.05.

Incubation of rat type II pneumocytes with C. pneumoniae was associated with an increased expression of TLR4 mRNA (Figure 1). The mRNA concentration for TLR4 roughly doubled after 2 hours of incubation with C. pneumoniae compared to non-infected controls. The ratio between TLR4 mRNA signal and GAPDH signal was 0.8 in control type II cells and 1.4 after 2 hours of treatment with C. pneumoniae.

In contrast, 2 hours of incubation did not affect TLR2 mRNA expression, the ratio of TLR2 mRNA and GAPDH signal was 1.3 in control and 1.1 in C. pneumoniae treated cells (Figure 1).

The density of the shown GAPDH bands was homogenous for both infected and non-infected type II cells. The second GAPDH band showed a 0.89-fold expression compared to the first band.

To test whether C. pneumoniae contact rapidly increases the TLR4 protein expression and whether newly synthesized TLR4 protein is required, we first characterized C. pneumoniae-mediated TLR4 protein expression time-dependent in isolated rat type II cells. The presence of TLR4-protein was confirmed by Western blotting in non-infected and in type II cells after 30, 60, 120 and 180 minutes incubation with C. pneumoniae (Figure 2A and 2B). As quantified by scanning the chemiluminescence's bands from four different experiments. The TLR4 protein expression was highest at 30 and 180 min (1.6-fold increase versus control cells). After 60 min and 120 min incubation with C. pneumoniae the TLR4 expression did not differ from non-infected cells. These two peeks suggest that after 30 min contact with C. pneumoniae TLR4 protein is expressed constitutively at a post-transcriptional level whereas 3 hours incubation with bacteria mediated newly synthesized TLR4 protein expression.

TLR4 protein was only detectable with a goat polyclonal anti-TLR4 antibody (L-14), mapping an epitope within the extra cellular domain.

Confocal imaging revealed rapid recycling of TLR4 from cytoplasm to the cell surface in response to C. pneumoniae incubation.

Binding-assays with C. pneumoniae incubation of type II cells demonstrated a TLR4 expression on the external membrane of the cells but not on non-infected cells (Figure 3A).

Time-dependent incubation with and without C. pneumoniae at 37°C, that was followed by fixation and permeabilization of the cells, revealed a TLR4 mobilization from the cytoplasm to the external cell surface that reached a peak value between 10–30 min after challenge with C. pneumoniae. The surface TLR4 expression started to decrease after 60 min in C. pneumoniae incubated cells. In non-infected cells TLR4 was partially co-localized with the intact F-actin cytoskeleton close to the cell membrane. A significant change in the F-actin cytoskeleton was found in cells after 30 and 60 minutes of C. pneumonia incubation (Figure 3B).

Western blot analysis was used to determine the influence of protein components and/or LPS of C. pneumoniae on the expression of TLR4 in type II pneumocytes (Figure 4A and 4B). Incubation of type II cells with C. pneumoniae for 30 min stimulated TLR4 expression 2.1-fold when compared with non-infected controls. The TLR4 expression was significantly lower after incubation of cells with heat-inactivated bacteria (56°C for 30 min) than after 30 min of treatment with viable C. pneumoniae. Pre-treatment of C. pneumoniae with polymyxin B (2 μg/ml) did not alter the TLR4 expression.

In experiments designed to block NF-κB activity, type II cells were pre-incubated with 20 μg/ml anti-TLR4 monoclonal antibodies (HTA125 or isotype control antibody) for 30 min and thereafter incubated with C. pneumoniae for 30 min. The effect on IκBα and NF-κB p65 was studied qualitatively (Figure 5A) and semi-quantitatively by CLSM. In non-infected type II cells IκBα was found in the cytoplasm and co-localized with the F-actin cytoskeleton. 30 min of bacterial contact caused a significant (p < 0.0001) decrease of the amount of IκBα in the cytoplasm when compared with control cells (15.9 ± 6 vs. 90.3 ± 16). Staining with anti-p65 antibody (recognizes p65 only when IκBα was not bound to p65) showed almost no activation of p65 in cytoplasm and nucleus in non-infected cells. Activated p65 was significantly (p < 0.0001) increased in the nucleus after C. pneumoniae contact (114 ± 10 vs. 6.3 ± 1.4 in control cells). Pre-treatment of cells with anti-TLR4 monoclonal antibodies, followed by C. pneumoniae incubation prevented IκBα decrease and p65 activation. Isotype control antibody had no effect on C. pneumoniae-mediated NF-κB activation (results not shown).

The amount of cytoplasmatic IκBα and of nuclear p65 was measured by Western blot analysis (Figure 5B and 5C). In comparison to control cells a 4-fold decrease in the amount of IκBα protein in the cytoplasm and a 10-fold increase of p65 in the nucleus was found 30 min after C. pneumoniae contact. In cells pre-treated with 20 μg/ml anti-TLR4 (HTA) antibodies, followed by C. pneumoniae incubation for 30 min neither C. pneumoniae-mediated IκBα degradation in the cytoplasm nor NF-κB activation of p65 in the nucleus was found.

As shown in Figure 6A, mRNA expression for TLR4 approximately doubled in type II cells exposed to C. pneumoniae for 3 hours compared to controls. The TLR4 mRNA concentration remained at baseline values in BAPTA-AM and SN50 pre-incubated type II cells followed by 3 hours incubation with C. pneumoniae. The ratio between TLR4 mRNA signal and GAPDH signal was 1.7 in control cells, 1.0 in C. pneumoniae treated cells, 1.6 in BAPTA-AM pre-treated and infected cells and 1.8 in parthenolide pre-treated and infected cells. The density of the shown GAPDH bands was homogenous with GAPDH bands showing a 0.91, 0.98 and 0.84 fold expression compared to the first band, respectively.

The amount of TLR4 protein was analyzed by Western blot (Figure 6B) and quantified by scanning the chemiluminescence's bands from three different experiments (Figure 6C). TLR4 protein expression exposed to C. pneumoniae for 3 hours was 2.5-fold higher than in control cells. In contrast, the TLR4 protein expression in BAPTA-AM and SN50 pre-incubated type II cells followed by 3 hours of incubation with C. pneumoniae remained at control levels.

Tables 1 and 2 show that C. pneumoniae stimulate the release of TNF-α and MIP-2 from type II cells in a time-dependent manner. TNF-α concentration in the supernatant increased after 3–6 hours incubation with bacteria, and remained constant over the next 12 hours. The MIP-2 concentration in the supernatant continuously increased after bacterial incubation. To rule out a stimulatory effect due to contamination by macrophages (isolated type II cells contain less than 10% macrophages), the release of TNF-α and MIP-2 from isolated macrophages obtained from rat bronchial lavage fluid was measured. As tables 1 and 2 show, an estimated contamination of 10% macrophages would not influence the TNF-α or MIP-2 concentration in the supernatant. Pre-incubation of cells with BAPTA-AM, parthenolide and SN50 suppressed TNF-α and MIP-2 production (Table 3).

To investigate whether C. pneumoniae-mediated TLR4 protein expression interferes with the F-actin-mediated Rho-GTPase activation, we examined the Rac1 and Rho protein expression after C. pneumoniae incubation for 30 min by CLSM. C. pneumoniae resulted in an increased expression of membrane-bound Rac1 and RhoA (Figure 7A). Furthermore, we tested the ability of mevastatin to down-regulate Rac1 and RhoA protein expression. As shown in Figure 7A mevastatin treatment reduced C. pneumoniae-mediated expression of both proteins. In non-infected type II cells, pre-treatment with mevastatin caused a destruction of the F-actin cytoskeleton (results not shown). We also observed that C. pneumoniae-induced stimulation of TLR4 can be decreased by mevastatin pre-treatment (Figure 7A).

Next, the Rac1, RhoA and TLR4 expression was examined by Western blotting of the membrane fractions of cells incubated with C. pneumoniae for 30 min in absence and presence of mevastatin (Figure 7B and 7C). As shown, C. pneumoniae resulted in a greater expression of Rac1 (1.5-fold), RhoA (3-fold) and TLR4 (2.5-fold) versus control cells. Mevastatin reduced C. pneumoniae-mediated Rac1, RhoA and TLR4 expression to control values.