The FASEB Journal • Research Communication
The antimicrobial peptide cathelicidin enhances activation of lung epithelial cells by LPS Renat Shaykhiev,*,1,2 Johannes Sierigk,*,1 Christian Herr,*,‡ Gabriela Krasteva,† Wolfgang Kummer,† and Robert Bals*,‡,3 *University Hospital Giessen and Marburg, Department of Internal Medicine, Division of Pulmonology, Universities of Giessen and Marburg Lung Center (UGMLC), Philipps University Marburg, Marburg, Germany; †Institute for Anatomy and Cell Biology, Excellence Cluster Cardio Pulmonary System, UGMLC, Justus Liebig University, Giessen, Germany; and ‡Department of Internal Medicine V–Pulmonology, Respiratory Intensive Care Medicine, Allergology, Homburg, Germany Epithelial cells (ECs) are usually hyporesponsive to various microbial products. Detection of lipopolysaccharide (LPS), the major component of gram-negative bacteria, is impeded, at least in part, by intracellular sequestration of its receptor, Toll-like receptor-4 (TLR4). In this study, using human bronchial ECs (hBECs) as a model of mucosal epithelium, we tested the hypothesis that the human LPS-binding, membrane-active cationic host defense peptide cathelicidin LL-37 augments epithelial response to LPS by facilitating its delivery to TLR4-containing intracellular compartments. We found that LL-37 significantly increases uptake of LPS by ECs with subsequent targeting to cholera toxin subunit B-labeled structures and lysosomes. This uptake is peptide specific, dose and time dependent, and involves the endocytotic machinery, functional lipid rafts, and epidermal growth factor receptor signaling. Cathelicidin-dependent LPS internalization resulted in significant increased release of the inflammatory cytokines IL-6 and IL-8. This indicates that, in ECs, this peptide may replace LPS-binding protein functions. In polarized ECs, the effect of LL-37 was restricted to the basolateral compartment of the epithelial membrane, suggesting that LL-37-mediated activation of ECs by LPS may be relevant to disease conditions associated with damage to the epithelial barrier. In summary, our study identified a novel role of LL-37 in host-microbe interactions as a host factor that licenses mucosal ECs to respond to LPS.— Shaykhiev, R., Sierigk, J., Herr, C., Krasteva, G., Kummer, W., Bals, R. The antimicrobial peptide cathelicidin enhances activation of lung epithelial cells by LPS. FASEB J. 24, 4756 – 4766 (2010). www.fasebj.org
ABSTRACT
Key Words: host defense 䡠 mucosal immunity 䡠 innate immunity 䡠 endotoxin 䡠 Toll-like receptor
cludes tight junctions, the mucociliary escalator, and antimicrobial peptides, epithelia of different organs are able to recognize microbes and respond to them by a production of an array of defense factors like proinflammatory cytokines and bactericidal substances (1). Therefore, ECs may be viewed as active players during host-pathogen interaction, not just targets of or passive barrier against microbes. Indeed, detection of bacteriaassociated patterns through epithelial pattern-recognition receptors such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain proteins (NODs) have recently been recognized as an important mechanism for the induction of local antimicrobial peptides, regulation of immune cell activation (2), and regulation of epithelial barrier integrity (3, 4). Lipopolysaccharide (LPS; also known as endotoxin), the major component of the outer membrane of gramnegative bacteria, is a potent activator of the innate immune system (5, 6). Interaction of LPS with TLR4 expressed by host cells leads to induction of proinflammatory mediators like TNF-␣, IL-1, IL-6, IL-8, costimulatory molecules that play a central role in the development of immune responses against LPS-containing bacteria (7). Being continuously exposed to the outside environment, ECs possess a number of mechanisms that protect them and, hence, the host defense system, in general, from permanent activation by microbial factors (1, 8). Some of these strategies are related to epithelial TLR signaling and include either relatively low steady-state expression or intracellular compartmentalization of components of the microbe-sensing complex (9). This may be exemplified by epithelial recognition of LPS that is considered to be less effective as compared to inflammatory cells of hematopoietic 1
Epithelial cells (ECs) form a continuous barrier against environmental exposures, including exposure to pathogens. In addition to physical segregation of the microorganisms and their inactivation by a multicomponent epithelial-specific defense apparatus that in4756
These authors contributed equally to this work. Current address: Department of Genetic Medicine, Weill Cornell Medical College, New York, NY, 10021, USA. 3 Correspondence: Department of Internal Medicine V–Pulmonology, Respiratory Intensive Care Medicine, Allergology, D-66421 Homburg, Germany. E-mail:
[email protected] doi: 10.1096/fj.09-151332 2
0892-6638/10/0024-4756 © FASEB
origin due to either lower expression of its receptor TLR4 and/or adaptor molecules MD-2 and CD14 on the EC surface (10) or intracellular localization of the TLR4. In the lung and intestinal epithelia, TLR4 has been found to reside within the Golgi apparatus (9, 11, 12), suggesting that a special LPS-transporting system may be necessary to activate epithelial TLR4. Inability to reach the receptor and activate TLR4 signaling may compromise the ability of the host to develop an appropriate antimicrobial response against LPS-containing bacteria (13). Antimicrobial peptides (AMPs) are endogenous effector molecules produced by the host defense cells, including ECs (14). Cathelicidins, along with defensins, represent the major family of AMPs in mammalian hosts, and LL-37/hCAP-18 is the only cathelicidin expressed in humans (15). The 37amino acid–long C terminus represents the antimicrobially active cationic peptide and is referred to as LL-37 (16). A growing body of evidence suggests that, in addition to its direct bactericidal activity, cathelicidin exerts various nonantimicrobial effects in host cells, including chemoattraction of neutrophils and T cells (17), regulation of dendritic cell (DC) maturation (18, 19), angiogenesis (20), and stimulation of epithelial repair (21, 22), activating various receptors, including epidermal growth factor receptor (EGFR) (23). As a cationic molecule, LL-37 is able to interact electrostatically with the negatively charged molecules, such as anionic lipid A portion of LPS (24). However, in contrast to LPS-binding protein (LBP), which delivers LPS to the TLR4 signaling complex and augments inflammatory responses to LPS (25), LL-37 decreases the effects of LPS on myeloid cells (26) and plays a protective role in sepsis (27, 28) by inhibition of the binding of LPS to LBP, and, consequently, to CD14⫹ cells (24). Along with its ability to neutralize LPS in monocytes and macrophages, LL-37 has been demonstrated to induce the cellular uptake of negatively charged nucleic acids such as plasmid (29) or autoreactive DNA (30), triggering in the latter case endosomal TLR9 activation in plasmacytoid DCs. Notably, LL-37 itself can also be internalized into ECs by an endocytosisdependent actin-independent mechanism (31). Since ECs are initial targets of microbial attacks and are frequently exposed to endotoxin, the aim of the study was to define how the LPS-binding host defense peptide LL-37 modifies epithelial responses to LPS.
MATERIALS AND METHODS Antibodies and reagents Ultrapure LPS from Salmonella minnesota was purchased from Alexis (Lausen, Switzerland), and Alexa Fluor 488labeled LPS from S. minnesota from Molecular Probes (Leiden, The Netherlands). The synthetic peptide LL-37 (N-LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTESCOOH) was purchased from Humboldt University (Berlin, CATHELICIDIN ENABLES ENDOTOXIN DETECTION
Germany). Polyclonal rabbit antibodies against human caveolin 1, clathrin, and TLR4 were obtained from Biozol (Echingen, Germany). Cy5-labeled mouse monoclonal antibody against human LAMP1 was purchased from Abcam (Cambridge, UK). The Alexa Fluor 647-labeled secondary goat anti-rabbit Ig antibody and the Alexa Fluor 647conjugated cholera toxin subunit B (CTxB) were purchased from Invitrogen (Eugene, OR, USA). To exclude nonspecific effects of LL-37, control experiments were performed with scrambled LL-37 (s-LL-37) (20). Antibodies against CD14 and TLR4 were purchased from Invivogen (Toulouse, France). Cell culture Primary human bronchial epithelial cells (hBECs) were isolated from large airways resected during lung explantation, as described previously (32). Received resections were macroscopically and microscopically cancer-free. The protocol was approved by the ethics committees of the Universities of Marburg and Munich (Dr. Ju¨rgen Behr, University of Munich, provided hBECs), and informed consent was obtained from donors. The cells were cultivated either as conventional submersed cultures or as air-liquid interface (ALI). Differentiation of the cells in ALI culture was controlled by measurement of the transepithelial resistance. NCI-H292 cells, a human pulmonary mucoepidermoid carcinoma cell line, was propagated at 37°C in 5% CO2 in RPMI 1640 medium (Life Technologies, Grand Island, NY, USA) supplemented with 2 nM l-glutamine and containing 1% penicillin/streptomycin. Analysis of LPS uptake NCI-H292 cells or hBECs were seeded at a density of 2.5 ⫻ 105 cells/well in a 12-well plate. For the experiments, serum-free conditions were achieved by omitting serum 24 h prior to the experiment. LL-37 and LPS were added to the cells at the indicated concentrations, depending on the experiment, ranging from 5 to 50 g/ml for LL-37 and 1 to 10 g/ml for LPS. For flow cytometry analysis of LPS uptake, Alexa Fluor 488-labeled LPS was used. After incubation as indicated, the cells were washed twice with ice-cold PBS, trypsinized, and suspended in cold growth medium, washed by centrifugation at 4000 rpm, and then subjected to FACS analysis (FACSort; BD Bioscience, San Jose, CA, USA). Colocalization studies with endocytotic markers NCI-H292 cells or hBECs were seeded onto 18-mm-diameter coverslips and incubated for 2 d. For detection of CTx-B, cells were fixed with 3.7% paraformaldehyde for 15 min at room temperature (RT). For detection of caveolin, cells were fixed with ⫺80°C methanol for 20 min. Following fixation and extensive washing, the cells were permeabilizied with 0.1% Tween in PBS for 30 min. After subsequent blocking with 5% BSA for another 30 min, the samples were incubated overnight at 4°C with CTxB (1:200) or antibodies against caveolin (1:500), and LAMP1 (1:100). Then, coverslips were washed with ice-cold PBS and probed with the Alexa-conjugated secondary antibodies for 1 h at room temperature. Coverslips were mounted in antifade (AppliChem, Darmstadt, Deutschland). Uptake of LPS was analyzed using a LSM 510 laser scanning microscope (Zeiss, Jena, Germany). Inhibition studies To determine the involvement of specific endocytotic pathways and mechanisms, in selected experiments, 1 h prior to 4757
stimulation with LPS and/or LL-37, cells were pretreated with the following inhibitors (all from Sigma, Munich, Germany, unless otherwise indicated): inhibitors of raft-dependent endocytosis genistein (200 M), methyl--cyclodextrin (MCD; 500 M) in combination with 250 ng/ml mevinolin (both exert membrane cholesterol-depleting activity); inhibitor of clathrin-dependent endocytosis chlorpromazine (5 g/ml); inhibitor of actin polymerization cytochalasin D (2 M; was added 30 min prior stimulation with LPS and/or LL-37); and specific inhibitor of EGFR tyrosine kinase tyrphostin AG1478 (10 M; A.G. Scientific, San Diego, CA, USA). Cell viability was monitored using the lactate dehydrogenase (LDH) cytotoxicity detection kit (Roche, Mannheim, Germany). Indicated concentrations of inhibitors were found not to be cytotoxic (data not shown). ERK inhibitor II FR180204 was purchased from Calbiochem (Darmstadt, Germany). Fluorescence resonance energy transfer (FRET) analysis Confocal laser scanning microscopy (CLSM) and FRET analysis served to identify spatial association between LPS and TLR4, modifying a technique described previously (33). pHECs were cultured on 8-well slides and incubated with LPS conjugated to Alexa 488 fluorophore (LPS/Alexa-488; at the indicated concentration) and LL-37 (at the indicated concentration) for 2, 4, 8, and 24 h. Control wells were incubated with vehicle instead of LPS. Cells were fixed with 4% paraformaldehyde (PFA) for 15 min and then incubated for 1 h with 10% heat-inactivated horse serum containing 0.1% BSA and 0.5% Tween 20 in 0.005 M (PBS). TLR4-antibody was diluted 1:50 in 0.005 M PBS containing 0.01% NaN3 and 4.48 g/L NaCl and applied to cells overnight at room temperature. After a washing step, Cy3-conjugated donkey anti-rabbit-Ig (1: 2000; Chemicon, Temecula, CA, USA) was applied for 1 h. Slides were rinsed, postfixed for 10 min in 4% PFA, rinsed again, and coverslipped with Mowiol 4 – 88 (pH 8.6; Merck, Darmstadt, Germany). Specificity of the secondary antibody was controlled for by omission of the anti-TLR4 antibody. FRET was detected by the acceptor photobleaching method using a CLSM (TCS-SP2 AOBS; Leica, Bensheim, Germany). Because of the overlapping absorbance spectra of Alexa-488 and Cy3, complete bleaching of the acceptor (Cy3) results in significant bleaching of the donor (Alexa-488) as well, so that we applied only partial acceptor bleaching. Acceptor (TLR4/ Cy3) fluorescence was bleached 5 times at maximum zoom (⫻32) with 100% laser power of the 543-nm HeNe laser line. Acceptor (TLR4/Cy3) fluorescence was bleached 5 times at maximum zoom (⫻32) with 100% laser power of the 543 nm HeNe laser line, resulting in mean reduction of fluorescence intensity of 21.5%. Compared to complete acceptor bleaching protocols, as we have applied earlier (33), smaller increases in donor fluorescence are recorded, and the probability to be unable to detect interactions increases. We kept this couple of fluorophores since it produced highest intensity of the individual signals, and the influence on sensitivity is conservative with respect to positive detection of FRET, i.e., it does not increase the rate of false-positive findings. Differences in the fluorescence intensity of the donor (LPS/Alexa488) were recorded in a defined area referred to as region of interest (ROI) before and after bleaching. To assess fluctuations in fluorescence that were not due to FRET, 4 other regions of interest (ROI 2-ROI 5) were analyzed adjacent to the bleached area. The CLSM settings were as follows: detection of FITC-10% laser power at 488, detection at 490 –540 nm; Cy3–50% laser power at 543 nm, detection at 550 – 625 nm. Photomultiplier tube (PMT) settings were between 480 and 550 to measure in the linear range of the PMT. FRET efficiency was recorded as change of fluorescence intensity of the donor (LPS/Alexa-488); ⌬IFdonor ⫽ IDA ⫺ IDB, where 4758
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IDA is the donor intensity after bleaching and IDB the donor intensity before bleaching. Background ⌬IF, defined as median value of ⌬IF recorded in nonbleached ROIs, was subtracted from ⌬IF measured in the bleached ROI, resulting in ⌬⌬IF representing the specific effect. Cytokine ELISA Cytokine levels in culture supernatants were determined using commercially available DuoSet ELISA Development kits for IL-6 and IL-8 (R&D Systems, Wiesbaden-Nordenstadt, Germany), according to manufacturer’s instructions. Statistical analysis Values are displayed as means ⫾ sd. Comparisons between groups were analyzed by ANOVA for experiments with ⬎2 subgroups. Post hoc range tests were performed with the t test (2-sided) with Bonferroni adjustment. Data from FRET-CLSM analysis were statistically evaluated by nonparametric KruskalWallis (k⬎2 groups) and Mann-Whitney U test (k⫽2). Results were considered statistically significant for P ⬍ 0.05.
RESULTS LL-37 increases uptake of LPS into ECs On the basis of the knowledge that LL-37 can bind LPS (24, 26); is able to transfer and retain negatively charged molecules such as DNA in intracellular compartments (29), triggering there TLR-dependent innate immune responses (30); and exhibits potent membranotropic activities and interferes with endocytotic machinery to enter ECs (31), we aimed to test whether interaction of LL-37 with LPS is capable of modulating the uptake of LPS into airway ECs. Human airway NCI-H292 cells were exposed to Alexa Fluor 488labeled LPS, and LL-37 was added at different concentrations. The uptake of LPS was determined by FACS. Whereas in the absence of LL-37, LPS is internalized into ECs at relatively low levels, the peptide significantly increased the uptake of LPS in a dose-dependent manner (Fig. 1A). We then asked whether such a mechanism is also active in primary airway ECs and found a similar effect of the peptide on LPS uptake (Fig. 1B). LL-37-dependent internalization of LPS into ECs was time dependent (Fig. 1C). These data provide evidence that cathelicidin augments the uptake of LPS into ECs. LL-37-mediated LPS uptake involves raft-dependent endocytosis and EGFR signaling Several pathways are involved in the uptake of extracellular material into cells, including actin-dependent phagocytosis and pinocytosis and actin-independent endocytosis via clathrin-dependent and caveolae/raftdependent pathways (34). Previous studies demonstrated that LL-37 can be internalized into ECs using an actin-independent endocytotic mechanism (31) and that uptake of LL-37-complexed DNA is associated with
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Figure 1. LL-37 induces uptake of LPS in lung ECs. A, B) NCI-H292 cells (A) and hBECs (B) were stimulated with 1 g/ml Alexa Fluor 488-labeled LPS in the absence or presence of LL-37 for 24 h. Intracellular accumulation of LPS was determined by FACS analysis and is shown as mean fluorescence intensity (n⫽3 in each group). LL-37 increases LPS uptake in a dose-dependent manner. C) NCI-H292 cells were stimulated with 1 g/m Alexa Fluor 488-labeled LPS in the presence of 20 g/ml LL-37, and intracellular accumulation of LPS was measured at indicated timepoints. Note that LL-37-dependent LPS uptake is time dependent. Data of a representative experiment are shown. ***P ⬍ 0.0001; n ⫽ 3.
lipid rafts (29), suggesting that LL-37 may utilize raftdependent endocytotic pathway to transfer negatively charged molecules into the cell. Lipid rafts are cholesterol-enriched lipid membrane microdomains implicated in and regulated by receptor tyrosine kinase signaling, and therefore sensitivity of endocytosis via these structures to nonacute cholesterol depletion with agents such as MCD and mevinolin, and tyrosine kinase inhibitors, such as genistein, distinguishes this endocytotic mechanism from both the clathrin-dependent and constitutive pinocytotic pathways (35). We applied different inhibitors of the raft-dependent endocytotic pathway and found that disintegration of lipid rafts by MCD and mevinolin and inhibition of tyrosine kinase activity by genistein significantly reduced the LL-37dependent uptake of LPS in NCI-H292 cells (Fig. 2A). Some activities of LL-37 are linked to functional EGFR (21,23). Because raft-dependent endocytosis can be triggered by an increase of tyrosine kinase activity (35) and inhibition of the latter reduced LL-37-mediated LPS internalization (Fig. 2A), we investigated whether activation of EGFR tyrosine kinase is relevant to the peptide’s effect on LPS uptake. Application of an inhibitor of EGFR-specific tyrosine kinase tyrphostin AG1478 resulted in a significant reduction of LL-37mediated LPS uptake (Fig. 2A). By contrast, pretreatment of cells with cytochalasin D, an inhibitor of actin-dependent endocytosis, did not decrease but increased the effect of LL-37 on LPS uptake (Fig. 2A), ruling out the involvement of phagocytotic and macropinocytotic routes of internalization. Application of chlorpromazine, a specific inhibitor of clathrin-dependent endocytosis, had less effect on LPS uptake as compared to other inhibitors (Fig. 2A). To exclude a nonspecific effect of the peptide due to its positive charge, we used a scrambled version of LL-37, called sLL-37, in which amino acid residues were randomly CATHELICIDIN ENABLES ENDOTOXIN DETECTION
replaced without changing the net charge of the molecule and found no effect on LPS uptake (Fig. 2B). This suggests that LPS targeting to the intracellular compartment is not solely based on the positive charge of LL-37, although this is essential for LPS binding. To test whether TLR4 is directly involved in the uptake of LPS in the presence of LL-37, we applied neutralizing antibodies to TLR4 and CD14 and also used polymyxin B. These interventions had no effect on the uptake of labeled LPS into ECs (Fig. 2C, D). Also, the surface expression of TLR4 on NCI-H292 cells after stimulation with LPS or LL-37 was not altered (data not shown). To investigate whether activation of ERK, a MAP kinase representing a downstream signaling element shared by EGFR and TLR4 pathways and previously shown to be involved in EGFR-dependent activation of epithelial cells by LL-37 (23), might be involved in the LPS uptake, we applied the ERK inhibitor. We did not detect an effect on LL-37-dependent LPS ingestion (data not shown). In summary, these data indicate that LL-37-mediated uptake of LPS does not require recognition of LPS by TLR4 and CD14. LL-37 targets LPS to a TLR4-containing intracellular compartment To gain an initial insight into the nature of uptake, we analyzed NCI-H292 cells by microscopy and found increased amounts of labeled LPS in vesicular cytoplasmatic perinuclear structures when the cells were exposed to LPS in the presence of LL-37 (Fig. 3A). To determine the cellular compartment to which LPS is targeted by LL-37, we performed colocalization experiments using markers for the Golgi apparatus (Alexa Fluor 647-labeled CTxB) and lysosomes (Cy5-labeled monoclonal antibody against LAMP1). CTxB also labels GM1 raft structures (36); at 4 h, LPS colocalizes only to 4759
Figure 2. Caveolae/lipid rafts, functional EGFR, and the native primary structure of LL-37, but not TLR4, are required for LL-37-dependent uptake of LPS. A) NCI-H292 cells were incubated with the indicated inhibitors 1 h prior to stimulation with LPS (1 g/ml) and LL-37 (10 g/ml). Inhibitors of raft-dependent endocytosis (genistein and MCD ⫹ mevinolin), as well as EGFR tyrosine kinase inhibitor tyrphostin AG1478, significantly reduced the LL-37-mediated uptake of LPS. Inhibition of clathrindependent endocytosis by chlorpromazine was less effective, and application of cytochalasin D increased LPS uptake. B) Effect of LL-37 depends on the primary structure of the peptide. NCI-H292 cells were stimulated with 1 g/ml LPS in the presence of 20 g/ml LL-37 or scrambled LL-37 (sLL-37) for 24 h at 37°C. sLL-37 showed no effect on the uptake of labeled LPS as measured by FACS analysis. Data of representative experiment are shown; n ⫽ 3. C) Neutralizing antibodies to TLR4 and CD14 were applied and did not inhibit, but rather increased, the LL-37-induced uptake of LPS. D) Application of polymyxin B did not inhibit the LL-37-induced uptake of LPS; n ⫽ 3. ***P ⬍ 0.0001.
the Golgi compartment. These are two morphologically and functionally distinct compartments of the endocytotic pathway (34) relevant to TLR4 signaling (12, 37). We found that after LL-37-induced uptake, labeled LPS colocalized with CTxB-labeled intracellular structures
after 4 h (Fig. 3B) and finally accumulated in LAMP1positive lysosomes after 24 h (Fig. 3C). There was no colocalization of labeled LPS with caveolin (stained with anticaveolin antibodies) and clathrin (stained with anticlathrin antibodies). Hence, these results indicate
Figure 3. Analysis LPS trafficking in ECs following LL-37mediated uptake. A) LL-37 (20 g/ml) augments the uptake of Alexa Fluor 488-labeled LPS (1 g/ml) into NCI-H292 cells. B, C) NCI-H292 cells were simultaneously incubated with Alexa Fluor 488-labeled LPS (1 g/ml) and LL-37 (10 g/ml) and then processed for confocal microscopy. Left micrographs show signal of the organelle markers (red), center micrographs show LPS signal (green), and right micrographs show merged images. Note that LPS is internalized into a perinuclear area. There is an accumulation of labeled LPS in the CTxB-positive Golgi apparatus after 4 h of incubation of cells with LPS in the presence of LL-37 (B). Labeling of lysosomes with Cy5-LAMP1 antibody (C) revealed a strong accumulation of LPS in lysosomes after 24 h of incubation. Scale bars ⫽ 50 m (A); 20 m (B, C). 4760
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that LL-37 induces the uptake of LPS into ECs, where the endotoxin is included in the Golgi apparatus before being delivered to the lysosomal compartment. The activation of TLR4 by LPS is a complex process and involves the interaction of several molecules, including CD14 and MD-2 (25). Previous studies have shown that, in ECs, TLR4 is localized to the Golgi apparatus (12,38), and activation of this receptor depends on LPS internalization and lipid raft integrity (38, 39). On the basis of the data that LL-37 increased delivery of LPS into the Golgi apparatus using a raftdependent mechanism, we proposed that this may lead to a physical interaction between endotoxin and its receptor, TLR4. To test this hypothesis, we performed FRET analysis with Alexa 488-labeled LPS and immunocytochemical detection of TLR4 with Cy3 as fluorophore in primary human bronchial ECs. After 2 h of incubation, Alexa 488-conjugated LPS was detected mostly in patches in association with the plasma membrane (Fig. 4A). The number of LPS patches increased with incubation time and was highest after 24 h of incubation. Also, the distribution pattern of labeled LPS dramatically changed at this latter time point with intensive diffuse accumulation in vesicular structures inside the cell. TLR4 immunoreactivity was located primarily diffusely in the cytosol and punctated in some areas of the cells. A highly significant (Pⱕ0.001) FRET between Alexa 488 (donor, conjugated to LPS) and Cy3 (acceptor, conjugated to the secondary antibody for TLR4 detection) was observed in a bleached area (region of interest, ROI 1) in cells after 4 h incubation with LPS and was also robustly detected after prolonged incubation times (8 and 24 h) (Fig. 4B). These results reveal a close spatial association between TLR4 and LPS, which occurs as early as 4 h after the first contact between the receptor and the bacterial toxin following LL-37-dependent LPS internalization. This is consistent with the colocalization of LPS with CTxB-labeled Golgi
at this time point (Fig. 3B). Thus, LL-37-mediated targeting of LPS to the Golgi apparatus via a raftdependent mechanism facilitates physical interaction of LPS with the intracellular TLR4 in ECs.
LL-37-dependent LPS uptake results in epithelial inflammatory response Recognition of LPS by TLR4 is associated with the transcription of proinflammatory genes (7). Whereas internalization of LPS by myeloid inflammatory cells is a critical step for its detoxification (40) and termination of TLR4-mediated inflammatory response (37), the functional consequences of LPS uptake into ECs are largely unknown but could be quite different since intracellular recognition of LPS is the only strategy to respond to endotoxin available for this cell type (9). Therefore, we analyzed whether augmentation of LPS uptake by LL-37 results in the release of increased amounts of inflammatory cytokines from ECs. Indeed, significantly higher levels of IL-8 were secreted by ECs after stimulation with LPS in the presence of LL-37 (Fig. 5A). The release of IL-6, another inflammatory cytokine, was also significantly increased (data not shown). This effect was dose dependent and, importantly, did not require serum, suggesting that LL-37 may replace otherwise essential serum-derived cofactors such as LBP. Moreover, LL-37-mediated EC response to LPS was dependent on LL-37-mediated LPS uptake via raft-mediated endocytosis pathway since application of nontoxic concentrations of genistein or MCD/mevinolin blocked the release of IL-8 (Fig. 5B). These data indicate that raft-mediated LL-37-induced uptake of LPS into ECs is associated with the release of proinflammatory cytokines.
Figure 4. FRET analysis of LPS-TLR4 interaction in hBECs. Cells were incubated with LPS-Alexa Fluor 488-fluorescence (donor) and TLR4-immunofluorescence (Cy3-conjugated secondary antibody, acceptor) 2, 4, 8, and 24 h after incubation with LPS in the presence of LL-37 (10 g/ml). Cy3 was bleached in the region of interest 1 (ROI 1). ROI 2–5, control areas outside the bleached area. Micrographs depict fluorescence intensity in false color representation (blue, low signal; yellow, high signal) before bleaching. Scale bar ⫽ 20 m. B) Changes in ⌬⌬IF in experimental (bl, bleached) compared to control ROIs. Median ⌬IF of each control group was arbitrarily set as 0. Box plots depict percentiles 0, 25, median, 75, and 100. Small circle and asterisk represent data outside 3 ⫻ sd, still included in the statistical analysis; n ⫽ 6 (control, 2 h) to 16 (bleached, 8 h). P values refer to nonparametric rank sum test (Mann-Whitney U test). In addition, ⌬⌬IF in bleached regions after 8 and 24 h of incubation was significantly higher (P⬍0.05) than after 2 and 4 h, respectively (Kruskal-Wallis test followed by Mann-Whitney U test). CATHELICIDIN ENABLES ENDOTOXIN DETECTION
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Figure 5. LL-37-mediated uptake of LPS is restricted to basolateral epithelial compartment and results in increased release of the proinflammatory mediator IL-8 from ECs. A) NCI-H292 cells were stimulated with 10 g/ml LPS in the presence or absence of LL-37 (5 or 10 g/ml). After 24 h of incubation, levels of IL-8 in cell supernatants were determined using ELISA. B) This inflammatory reaction was prevented when inhibitors of raft-dependent endocytosis genistein (200 M) and MCD (500 M)/mevinolin (250 pg/ml) were added for 1 h prior to stimulation with LPS (10 g/ml) and LL-37 (10 g/ml). C) LL-37-induced LPS uptake requires interaction with basolateral epithelial membrane compartment. LL-37 and Alexa Fluor 488-labeled LPS were applied to either the apical or basolateral side of differentiated hBECs. After 24h of incubation, intracellular accumulation of LPS was determined by FACS analysis. D) Basolateral, but not apical, application of LPS and LL-37 at indicated concentrations significantly increased release of IL-8 from differentiated hBECs. *P ⬍ 0.05; ***P ⬍ 0.0001; n ⫽ 8.
LL-37-mediated LPS-uptake is restricted to the basolateral membrane compartment Epithelial tissues are characterized by an apical-basolateral polarization with tight junctions that physically and functionally separate the outside environment from the tissue interior, as well as segregate apical and basolateral membrane domains (41). Since responses of polarized ECs to stimulation with certain microbial patterns are restricted to specific membrane compartment (42), we investigated whether the effect of cathelicidin on LPS uptake by polarized ECs and subsequent development of epithelial inflammatory response depend on the site of application. Differentiated airway ECs cultured in a 3-dimensional air-liquid interface transwell system (32) were stimulated with LPS with or without LL-37 from the apical or the basolateral side. No significant uptake of labeled LPS into ECs (Fig. 5C) and, consistently, no increase of the inflammatory cytokine release were detected following apical application of the stimuli (Fig. 5D). By contrast, basolateral application of LPS and LL-37 significantly increased LPS internalization and the release of IL-8 from differentiated ECs (Fig. 5D). These data suggest that LL-374762
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mediated activation of polarized ECs by LPS is restricted to the basolateral membrane compartment.
DISCUSSION ECs form a barrier between the inside and the outside of the body and, therefore, are frequently exposed to microorganisms present in the environment. On the one hand, ECs must provide a fast and effective host defense against pathogens to keep the body free from infection. On the other hand, ECs must avoid an exaggerated inflammatory response to the continuous microbial stimulation and maintain a stable barrier. Such balanced contribution of ECs to mucosal immunity is achieved, in part, through intracellular compartmentalization of microbe-sensing molecules, such as TLRs, allowing their activation and subsequent induction of inflammatory defense response by invasive pathogens while sequestering these receptors from noninvasive luminal bacteria (9). Recognition of LPS, a major microbial pattern associated with gram-negative bacteria, is an example for such segregated epithelial response. In ECs, TLR4 is predominantly localized to
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the intracellular compartments, such as the Golgi apparatus (11, 12), providing a mechanism for a steadystate hyporesponsiveness of ECs to endotoxin. LPS should be delivered to TLR4-enriched intracellular compartments to be sensed by ECs and induce innate immune activation. How this process is regulated remained obscure. The main finding of the present study is identification of an endogenous regulatory mechanism that enables ECs to sense and respond to LPS by increasing its internalization and subsequent interaction with TLR4 inside the cell. Two conditions are required for proinflammatory activation of ECs by LPS: the presence of the endogenous cationic host defense peptide cathelicidin LL-37, and the basolateral localization of both the peptide and endotoxin. Subsequent to the peptide-induced uptake, endotoxin is delivered to the proximity of the TLR4-complex followed by recognition and cell activation. The putative route of LL-37dependent LPS uptake includes several steps as part of the classic endocytotic process. The first event is crossing the membrane, i.e., internalization, since there is very little or no LPS in the cytoplasm of cells not stimulated with LL-37 (Fig. 3A). The second step is most likely targeting to the Golgi apparatus, since membrane raft-dependent endocytosis machinery (confirmed in inhibitory studies, Fig. 2A) appeared to be similar to that utilized by the cholera toxin. Four hours after stimulation, LPS was detected in cholera toxin-stained intracellular structures (Fig. 3B). Finally, LPS is further delivered to the lysosome compartment, which in the majority of cases represents the “late” (or terminal) step of intracellular traffic. At the later time points, colocalization of LPS with the lysosome marker LAMP1 was observed (Fig. 3C). The FRET data indicate physical interaction between LPS and its receptor, as it is necessary for TLR4-dependent recognition of endotoxin. LL-37 dependent uptake of LPS appears to be independent from classical components of LPS recognition, such as TLR4, CD14, and MD2. Inhibitory antibodies to TLR4 and CD14, as well as application of polymyxin B had no effect in LPS uptake, MD2 expression is not typical for human airway ECs (43). LL-37 is a short cationic peptide with membrane-active physical properties (16). Because of its cationicity, the peptide interacts with negatively charged molecules, such as DNA, mucin (44), and glycosaminoglycans (45). LL-37 also transfers complexed negatively charged molecules into cells. LL-37-mediated internalization of plasmid DNA has been used to transfect eukaryotic cells (29). The uptake of selfDNA complexed with LL-37 into DCs triggers activation of endosomal TLR9, an effect that has been implicated in the pathogenesis of lupus erythematosus (30). LL-37 binds to LPS (26) through electrostatic interaction with the anionic lipid A portion of this microbial product (24). However, by contrast to the well-documented LPS-neutralizing effect of cathelicidin (24, 26) and inhibition of TLR4 responses by this peptide in myeloid cells (19, 46), the data of the present study show that, in ECs, interaction between the peptide and endotoxin has different functional outcomes. As a host defense peptide, LL-37 exhibits a number of immunomoduCATHELICIDIN ENABLES ENDOTOXIN DETECTION
latory functions, including the ability to increase cytokine release from various cell types. In contrast, LL-37 can suppress inflammation by neutralizing inflammatory responses of myeloid cells, primarily monocytes, macrophages, and dendritic cells, to microbial components, including LPS. It is rather intriguing that, in ECs, that are usually hyporesponsive to microbial stimuli, including LPS, LL-37 can potentiate innate immune responses using an additional pathway identified in the present study, i.e., by increasing uptake of LPS, enabling its interaction with its receptor TLR4 and subsequent inflammatory cytokine release. The differences in receptor composition and distribution between epithelial and myeloid cells seem to determine the character of LL-37LPS interaction. LL-37 is toxic to cells at high concentrations: ⬎30 g/ml; concentrations necessary for the effect on LPS uptake were not associated with damage of ECs (21). The concentrations used in this study are relevant to the in vivo levels that have been detected to range from 1 to 1000 g/ml in airways secretions (47, 48). We found that LL-37 facilitates internalization of LPS into ECs via a raftdependent endocytosis pathway targeting this microbial product to the TLR4-containing intracellular compartments, where LPS physically interacts with its receptor TLR4. Consistent with our findings, a lipid-raft-dependent mechanism has been found critical for LL-37-mediated transfer of DNA into the host cells (29). Both LL-37 (31) and LPS (49) have previously been shown to use endocytotic machineries to enter ECs. The present study extends these observations and provides the first evidence that LL-37-dependent LPS internalization can be used by human primary mucosal ECs to augment their innate immune responsiveness to endotoxin. In this regard, LL-37 may be viewed as a natural analog of synthetic cell-penetrating peptides (CPPs) that can bind to various anionic molecules and transport them into the cell (50). The peptide-induced uptake of LPS is dependent on the specific structure of the peptide and not solely on its charge, since the scrambled version of the peptide had no effects, suggesting the possibility that the peptide may interact with a specific receptor to initiate the uptake of LPS. Indeed, several receptors have been described as mediators of LL-37’s effects on host cells, including FPRL1, as a direct receptor (17) and EGFR, as a signaling intermediate (21, 23). Since EGFR activation has been implicated in the recruitment of various endocytic molecules to lipid rafts (51) and selective inhibition of EGFR, tyrosine kinase was sufficient to block the effect of LL-37 on LPS uptake into ECs. LL-37-initiated transactivation of EGFR seems to be critical for the raft-dependent uptake of LPS mediated by the peptide. Application of an inhibitor of ERK1/2 did not modulate the LL-37-dependent uptake of LPS, indicating that the possibility a direct activation of ERK by a cathelicidins-LPS complex, acting as a positive feedback loop to increase the uptake of more peptide, is unlikely. The present study suggests a model, in which the interaction of LL-37 with LPS redirects the latter from the “classical” membraneassociated TLR4-CD14-dependent mechanism, which is active in myeloid cells but silent in ECs, to a “nonclassical” EGFR-dependent host defense pathway, which is independent from early TLR4/CD14-mediated LPS recognition and most likely unique to ECs. 4763
Another important finding of the present study is that in differentiated epithelium, the effect of LL-37 on LPS uptake and proinflammatory activation of ESs requires interaction with the basolateral compartment of the epithelial membrane. The hyporesponsiveness of ECs to stimulation by PAMPs is well recognized and thought to be necessary to avoid constant activation of cells that reside at body surfaces (52). Differentiated ECs are intrinsically polarized with various receptors being asymmetrically distributed between the apical and basolateral plasma membrane compartments. Importantly, many microbe-sensing receptors display low expression at the apical epithelial surface. A receptor for bacterial flagellin TLR5 is expressed on the basolateral, but not apical, surface of intestinal ECs (42), providing a mechanism that prevents inflammation unless the junctional barrier is damaged. TLR4 appears to be less expressed at the apical surface of the epithelium (53) consistent with its intracellular localization as shown previously (11, 12) and confirmed in our study for the primary human airway epithelium. Also, the signaling consequences after the activation of patternrecognition receptors depend on the site of expression and stimulation. Activation of TLR9 at the apical surface results in prevention of NF-B activation, whereas basolateral TLR9 signaling results in degradation of its inhibitor IB␣ and, subsequently, NF-B activation (54). Interestingly, expression of receptors of the EGFR family, which are involved in the effect of LL-37 on LPS uptake by ECs in our study, is also restricted to the basolateral epithelial compartment (55, 56). Epithelial wound repair processes depend on the segregated distribution of EGFR receptors (55). We used scrambled peptide to exclude an effect that is mainly due to charge. Because we saw no effect of the scrambled LL-37 on LPS internalization (Fig. 2B), we concluded that the conformation of the peptide is important for the effect, suggesting that a receptor-mediated mechanism is possible. Strong membrane staining for EGFR within the human bronchial epithelium, particularly evident between basal cells and the basal aspect of columnar cells, has been previously described (56). In our study, we found that basolateral application of LL-37 and LPS results in a stronger LPS uptake and IL-8 release from ECs. Consistent with this general mechanism to keep sensors of infection and injury segregated from environmental microbes and other danger signals unless the barrier is broken, LL-37mediated uptake of LPS into ECs is compartmentalized and requires interaction with the basolateral membrane domain. Exposure to LPS together with LL-37 from the apical side is not capable of fully activating ECs. In contrast, application to the basolateral side induces the release of proinflammatory cytokines. This effect of the peptide may be particularly relevant to microbe-host interactions associated with the damage to epithelial tight junctions. In case of gram-negative bacteria, LPS delivered to the basolateral space can be readily complexed with LL-37 secreted by ECs. Using the LL-37-dependent mechanism, LPS enters ECs 4764
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where it can interact with TLR4 and initiate a protective inflammatory response. Indeed, many pathogenic bacteria can damage the epithelial junctional barrier (57). In addition, this mechanism may be relevant to disease conditions characterized by mucosal inflammation. In the latter scenario, inflammatory cytokines can disrupt junctional integrity providing access for luminal LPScontaining bacteria to the basolateral compartment, while neutrophils recruited to the site of infection may supply additional amounts of LL-37 (58). In both scenarios, induction of epithelial inflammatory response by LPS requires LL-37 and interaction with the basolateral epithelial membrane. In summary, we identified a novel mechanism that enables ECs to detect and respond to LPS. This mechanism depends on the cathelicidin peptide LL-37 and the presence of both molecules, LPS and LL-37, at the basolateral surface of the epithelium. A novel function of the cathelicidin LL-37 revealed in this study—targeting LPS into TLR4-enriched intracellular epithelial compartments—is directly related to host-microbe interactions and likely relevant to antimicrobial host defense but is independent from well-known antimicrobial properties of the peptide. Invasive microorganisms, damage to the epithelial barrier or leakage of the tight junction system is required to enable LPS to access the basolateral compartment. The principal mechanism of the antimicrobial effect of LL-37 is penetration of the bacterial cell membrane. Although electrostatic interaction between LL-37 (cationic) and LPS (lipid A portion, anionic) is a prerequisite for LL-37 targeting of the majority of gram-negative bacteria, interaction with LPS alone leads to the neutralization of the latter, a mechanism that provides protection from sepsis, or, as it was revealed in our study, potentiates innate immune defense of cells that exhibit relatively low constitutive proinflammatory activity. As mentioned above, bactericidal effect and neutralization of LPS are largely charge-dependent, whereas modulation of host cell function, such as via facilitated LPS delivery to intracellular compartments sensitive to this microbial component, likely represents regulated receptormediated process. Several diseases involving mucosal organs are associated with a breakdown of epithelial integrity, and inadequate stimulation of immunity, including inflammatory bowel disease, asthma, cystic fibrosis, chronic obstructive lung disease, and cancer. LL-37 is known to be secreted by a number of cell types in these conditions and, based on the present results, could augment and perpetuate the chronic inflammatory processes. Conversely, defects in the mechanism revealed in the present study may result in impaired detection of endotoxin by epithelia and, consequently, infectious disease due to inability of ECs to sense and respond to LPS-containing pathogenic bacteria. The authors thank Prof. Dr. Ju¨rgen Behr (University of Munich, Munich, Germany) for providing ECs and Thomas Damm and Annette Pu¨chner for excellent technical assis-
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tance. This study was funded by the Bundesministerium fu¨r Bildung und Forschung (PROGRESS, C1; ASCONET/ COSYCONET) to R.B., by the Deutsche Forschungsgemeinschaft (DFG; Ba 1641/8 and SFB/TR 22 A8) to R.B., and by the Excellence Cluster CardioPulmonary System (DFG) to W.K. G.K. was supported by a postdoctoral stipend from the DFG Excellence Cluster CardioPulmonary System and a young scientist’s grant from the Medical Faculty of the Justus-Liebig-University (Giessen, Germany).
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Received for publication November 24, 2009. Accepted for publication July 29, 2010.
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