Chlamydophila pneumoniae induces expression of toll-like receptor 4 and release of TNF-alpha and MIP-2 via an NF-kappaB pathway in rat type II pneumocytes

Abstract

Background: The role of alveolar type II cells in the regulation of innate and adaptive immunity is unclear. Toll-like receptors (TLRs) have been implicated in host defense. The purpose of the present study was to investigate whether Chlamydophila pneumoniae (I) alters the expression of TLR2 and/orTLR4 in type II cells in a (II) Rho-GTPase- and (III) NF-kappaB-dependent pathway, subsequently (IV) leading to the production of (IV) pro-inflammatory TNF-alpha and MIP-2.

Methods: Isolated rat type II pneumocytes were incubated with C. pneumoniae after pre-treatment with calcium chelator BAPTA-AM, inhibitors of NF-kappaB (parthenolide, SN50) or with a specific inhibitor of the Rho-GTPase (mevastatin). TLR2 and TLR4 mRNA expressions were analyzed by PCR. Activation of TLR4, Rac1, RhoA protein and NF-kappaB was determined by Western blotting and confocal laser scan microscopy (CLSM) and TNF-alpha and MIP-2 release by ELISA.

Results: Type II cells constitutively expressed TLR4 and TLR2 mRNA. A prominent induction of TLR4 but not TLR2 mRNA was detected after 2 hours of incubation with C. pneumoniae. The TLR4 protein expression reached a peak at 30 min, began to decrease within 1-2 hours and peaked again at 3 hours. Incubation of cells with heat-inactivated bacteria (56 degrees C for 30 min) significantly reduced the TLR4 expression. Treated bacteria with polymyxin B (2 mug/ml) did not alter TLR4 expression. C. pneumoniae-induced NF-kappaB activity was blocked by TLR4 blocking antibodies. TLR4 mRNA and protein expression were inhibited in the presence of BAPTA-AM, SN50 or parthenolide. TNF-alpha and MIP-2 release was increased in type II cells in response to C. pneumoniae, whereas BAPTA-AM, SN50 or parthenolide decreased the C. pneumoniae-induced TNF-alpha and MIP-2 release. Mevastatin inhibited C. pneumoniae-mediated Rac1, RhoA and TLR4 expression.

Conclusion: The TLR4 protein expression in rat type II cells is likely to be mediated by a heat-sensitive C. pneumoniae protein that induces a fast Ca2+-mediated NF-kappaB activity, necessary for maintenance of TLR4 expression and TNF-alpha and MIP-2 release through possibly Rac and Rho protein-dependent mechanism. These results indicate that type II pneumocytes play an important role in the innate pulmonary immune system and in inflammatory response mechanism of the alveolus.

Figure 1

TLR4 mRNA expression in type II cells. Detection of TLR4 (upper lane) and TLR2 mRNA expression (middle lane) in type II cells after exposure to C. pneumoniae for 2 hours (1) and in non-infected control cells (2) by semiquantitative polymerase chain reaction. GAPDH (lower lane) served as internal control. After 2 hours of incubation with C. pneumoniae TLR4 mRNA expression in type II cells approximately doubled whereas TLR2 mRNA remained unchanged.

Figure 2

TLR4 protein expression in type II cells has two peeks. (A) The cytoplasm protein fraction of type II cells was subjected to SDS-PAGE and immunoblotting, non-infected cells (lane 1), 30 min (lane 2), 60 min (lane 3), 120 min (lane 4) and 180 min (lane 5) exposure with C. pneumoniae. (B) The TLR4 protein expression was estimated by densitometry. Values of n = 4 experiments are given as means ± SD in arbitrary units [AU]). Asterisk indicates a significant difference to controls (1), 60 min (3) and 120 min (4) (P <0.05).

Figure 3

TLR4 is localized extra-and intracellularly in presence of C. pneumoniae. (A) Representative CLSM images after binding of C. pneumoniae at type II cells. Cells were without permeabilization stained with anti-TLR4 antibody that was detected by a secondary antibody coupled to Alexa488 (green). TLR4 is expressed at the external cell membrane. In un-infected cells no TLR4 expression was seen. Transmission images with pseudo-color red was used to highlight the outline of the cells. (B) Representative CLSM images of type II cells after incubation with C. pneumoniae at 37°C. Cells were after permeabilization stained with anti-TLR4 antibody that was detected by a secondary antibody coupled to Alexa594 (red) and labeled for F-actin with Alexa488-conjugated phalloidin (green). Nuclear DNA was stained with DAPI (blue). TLR4 mobilization to the external cell membrane reached a peak value after 10–30 min after exposure to C. pneumoniae. Areas of overlap between red and green are yellow, the bar equals 10 μm. Shown are representative examples, three more experiments gave similar results.

Figure 4

TLR4 expression is stimulated by a heat-sensitive component of C. pneumoniae. (A) TLR4 was visulalized by Western blot technique and (B) the expression determined by densitometry in non-infected cells (lane 1), non-treated C. pneumoniae (lane 2), heat-inactivated C. pneumoniae (lane 3) and polymyxin pre-treated bacteria (lane 4). Values of n = 4 experiments are given as means ± SD in arbitrary units. Asterisk indicates a significant difference to non-treated C. pneumoniae (2) and polymyxin pre-treated bacteria (4) (P < 0.0001).

Figure 5

Inhibition of C. pneumoniae-mediated NF-κB activation with anti-TLR4 antibodies. (A) Representative CLSM images after incubation of type II cells pre-treated or not with anti-TLR4 antibodies followed by C. pneumoniae incubation for 30 min. Cells were stained with anti-IκBα antibody and anti-p65 antibody both detected by a secondary antibody coupled to Alexa594 (red), F-actin was stained with Alexa488-conjugated phalloidin (green) and nuclear DNA with DAPI (blue). In C. pneumoniae infected cells IκBα is decreased and p65 is localized in the nucleus. In cells that were treated with anti-TLR4 antibodies IκBα is not decreased and p65 is not localized in the nucleus. The effect on IκBα and NF-κB p65 was studied semi-quantitatively by CLSM as described in Materials and Methods. The C. pneumoniae-mediated IκBα degradation resulting in a 5.6-fold decrease versus control cells and NF-κB p65 translocation in the nucleus resulting in a 18-fold increase versus control cells. Areas of overlap between blue and red are pink, and between red and green are yellow, the bar equals 10 μm. (n = 5 experiments). (B and C) The amount of cytoplasmatic IκBα and of nuclear p65 of type II cells in controls (lane 1), incubated with C. pneumoniae for 30 min (lane 2) and pre-incubated with anti-TLR4 (HTA) antibodies prior to C. pneumoniae exposure for 30 min (lane 3) was quantified by Western blot analysis. Values of n = 3 experiments are given as means ± SD in arbitrary units. Open bars, IκBα; solid bars, p65. Asterisk indicates a significant difference to controls (1) and cells pre-incubated with anti-TLR4 antibodies (3) (P < 0.0001).

Figure 6

BAPTA-AM, parthenolide and SN50 inhibited C. pneumoniae-mediated TLR4 mRNA and protein expression. (A) Detection of TLR4 (upper lane) and GAPDH mRNA expression (lower lane) in type II cells by semi-quantitative polymerase chain reaction. The C. pneumoniae-induced TLR4 mRNA increase (2) returned to control values (1) after combined exposure to BAPTA-AM (3). Parthenolide (4) exerted an even stronger inhibitory activity and reduced TLR4 mRNA expression below baseline values. (B) Type II cells in suspension controls (lane 1) incubated with C. pneumoniae for 3 hours (lane 2), pre-incubated with BAPTA-AM (lane 3) and SN50 (lane 4) prior to C. pneumoniae exposure were prepared for Western blot analysis of TLR4 protein. (C) Their protein expression was determined by densitometry. Values of n = 3 experiments are given as means ± SD in arbitrary units. Asterisk indicates a significant difference to control (1), BAPTA-AM (3) and SN50 (4) (P < 0.05).

Figure 7

Mevastatin inhibited C. pneumoniae-mediated Rac1, RhoA and TLR4, expression. (A) Representative CLSM images of type II cells pre-treated and non-treated with mevastatin followed by C. pneumoniae incubation for 30 min. Detection with antibody for Rac1, RhoA and TLR4 followed by Alexa594-labeled secondary antibody (red). Labelled for F-actin with Alexa488-conjugated phalloidin (green) and nuclear DNA with DAPI (blue). The red labeled Rac1, RhoA and TLR4 appear yellow when co-localized with green labeled F-actin. The bar equals 10 μm. (B) Rac1, RhoA and TLR4 were visualized by Western blot technique and (C) their expression in membrane fractions was determined by densitometry in control cells (lane 1), C. pneumoniae incubated cells for 30 min in absence (lane 2) and in presence of mevastatin (lane 3). The values are means ± SD in arbitrary units of n = 3 experiments. Open bars, Rac1; solid bars, RhoA; hatched bars, TLR4. Asterisk indicates a significant difference compared to controls (1) and mevastatin (3) (P < 0.05).