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Inhibition of LPS-induced activation of alveolar macrophages by high concentrations of LPS-binding protein
BBRC Biochemical and Biophysical Research Communications 295 (2002) 553–560 www.academicpress.com
Inhibition of LPS-induced activation of alveolar macrophages by high concentrations of LPS-binding protein Lutz Hamann,a,b,* Cordula Stamme,c,d Artur J. Ulmer,a and Ralf R. Schumannb a Department of Immunology and Cell Biology, Center for Medicine and Bioscience, Borstel, Germany Institute of Microbiology and Hygiene, Charit e, Humboldt University Berlin, Dorotheenstraße 96, 10117 Berlin, Germany Department of Immunochemistry and Biochemical Microbiology Research Center Borstel, Center for Medicine and Bioscience, Borstel, Germany d Department of Anesthesiology, University of L€ubeck, L€ubeck, Germany b
c
Received 10 June 2002
Abstract Lipopolysaccharide (LPS)-binding protein regulates the effects of LPS on immunocompetent cells. By catalyzing the binding of LPS to membrane CD14, LPS-binding protein (LBP) potentiates both the inflammatory response and internalization of LPS. LBPmediated transport of LPS into high density lipoprotein particles participates in LPS clearance. Elevated serum levels of LBP have been shown to elicit protective effects in vivo. Because the expression of LBP is upregulated in lung epithelial cells upon proinflammatory stimulation, we here investigated whether LBP modulates inflammatory responses by lung specific cells. The moderate elevation of LBP concentrations enhanced both LPS-induced signaling and LPS uptake by rat alveolar macrophages, whereas strongly elevated LBP levels inhibited both. In contrast, the lung epithelial cell line A549 responded to high concentrations of LBP by an enhanced LPS uptake which did not result in cellular activation, suggesting an anti-inflammatory function of these cells by clearing LPS. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Inflammation; LPS-induced signaling; LPS uptake; Innate immunity; Alveolar macrophages; Lung epithelial
Lipopolysaccaride (LPS), the major cell wall component of Gram-negative bacteria, is a strong activator of the innate immune system triggering the release of various proinflammatory mediators, among them cytokines such as TNF-a, IL-1b, and IL-6 [1–3]. The effects of LPS on immunocompetent cells are strongly regulated by LPSbinding protein (LBP) in different ways. The most prominent feature of LBP is to catalyze the binding of LPS to the LPS receptor mCD14 by releasing LPS monomers from LPS micells and transfering them to mCD14 [4–6]. LPS binding to mCD14, mainly expressed on monocytes and macrophages, initiates a signaling cascade via toll-like receptor 4 and MD-2 [7,8]. Immune cells that do not express mCD14, such as endothelial cells and smooth muscle cells, can be stimulated by LPS to release cytokines via soluble CD14 (sCD14) [9]. The crucial role of LBP in recognizing LPS as well as whole bacteria has been shown by employing LBP knock out *
Corresponding author. Fax: +49-30-45052-24904. E-mail address: [email protected] (L. Hamann).
mice. Blood cells from LBP)/) mice do not release TNFa in response to LPS [10]. In vivo, LBP)/) mice fail to combat a Gram-negative infection with Salmonella typhimurium [11]. However, a dysbalanced release of these mediators triggers the onset of sepsis or septic shock [12]. Binding to mCD14 also results in LPS internalization and subsequent detoxification of LPS by professional phagocytes [13,14]. The internalization of LPS has been attributed to cells of the myeloid lineage, mainly monocytes and macrophages, due to the restricted expression pattern of mCD14 by these cells. On the other hand, LBP is associated with high density lipoproteins (HDL) particles and LPS-binding to LBP can result in LPS clearance via HDL particles [15]. In acute phase situations, the LBP expression by hepatocytes, which are considered to be the main source of LBP [16], is strongly upregulated by proinflammatory cytokines reaching serum levels of about 200 lg=ml [17]. We have recently shown that these concentrations of LBP have protective effects in a mouse model of bacteriemia and sepsis [18] and that high LBP concentrations can
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inhibit LPS induced TNF-a production by human monocytes [19]. Furthermore, we have shown that enhanced LBP levels mediate enhanced LPS uptake by Chinese hamster ovary cells without cellular activation which may be a mechanism for LPS clearance [20]. Recently, Dentener and colleagues [21] have documented that human respiratory type II cells are able to express LBP in response to IL-1b and IL-6 both of which might be released in vivo by LPS-stimulated alveolar macrophages. LBP concentrations of 100 ng/ml have been detected in broncho alveolar lavage (BAL) of patients with bacterial pneumonia [22]. Assuming that BAL dilutes alveolar fluids by 50–100-fold LBP concentrations should be much higher. The function of this elevated LBP expression is not known. Martin and coworkers [23] have shown in vitro that an increase of LBP concentration up to 100 ng/ml results in an enhanced activation of alveolar macrophages by LPS. However, due to the fact that the local expression of LBP by the lung epithelium is inducible by proinflammatory cytokines, LBP concentrations may reach significantly higher levels in the pulmonary compartment under proinflammatory conditions. It might be assumed that this locally increased LBP production results in a similar inhibition of LPS-induced signaling as it has been shown for elevated LBP concentrations employing a mouse model of sepsis and bacteriemia [18]. In the current study, we show that the effect of LBP on LPS uptake and LPS-induced signaling significantly varies with both the cell types, i.e., alveolar macrophages or type II cells, and the LBP concentration used. In rat alveolar macrophages, LBP concentrations up to 100 ng/ ml resulted in a dose-dependent increase of both LPS uptake and LPS-induced signaling. Further increase of the LBP concentration resulted in a remarkable inhibition of LPS-induced signaling and LPS uptake by these cells. In contrast, A549 cells, a respiratory type II cell line, showed an increase in LPS uptake by increasing LBP concentrations. The LBP-mediated increase of LPS uptake by A549 cells did not result in cellular activation, was found to be significantly inhibited by cytochalasin D, and was partially dependent on mCD14. Therefore, LBP concentrations higher than 100 ng/ml may result in the inhibition of LPS-induced signaling in vivo by either of two mechanisms. First, decrease of cellular association of LPS associated with an inhibited LPS-induced signaling by alveolar macrophages. Second, increase of an competitive LPS uptake by LPS unresponsive cells which may be a new mechanism of LPS clearance.
Linz, Austria), penicillin (100 U/ml), and streptomycin ð100 lg=mlÞ. The cells were grown at 37 °C in an atmosphere containing 5% CO2 . Alveolar macrophages were isolated by lung lavage of specific pathogen-free, male Sprague–Dawley rat weighing 250–300 g. Rats were anesthetized with pentobarbital and killed by exsanguination. The lungs were removed and lavaged six times with a macrophage isolation buffer containing 140 mM NaCl, 6 mM glucose, 2.5 mM phosphate buffer, 10 mM HEPES, and 0.2 mM EGTA, followed by two lavages with the same buffer containing 1.3 mM magnesium and 2 mM calcium and no EGTA. Samples were centrifuged at 200g for 10 min. Cell recovery routinely averaged 5–7 106 cells/animal. The viability of the cells was determined by erythrosin B exclusion and averaged 95–98%. Flow cytometry. Cell surface expression of mCD14 and TLR-4 was determined by flow cytometry using mAb’s Mem18 against CD14 (kindly provided by V. Horesji, Prague, Cezch Republic), HAT 125 against TLR-4 (kindly provided by K. Myake, Saga, Japan) and goat anti-mouse FITC as secondary antibody (Dianova, Hamburg, Germany). Cell surface binding of Re F515 LPS from the mutant Escherichia coli strain F515 (kind gift of H. Brade, Borstel, Germany) was assessed by immunofluorescence employing the anti-Re F515 LPS mAb clone A25 [24], and goat anti-mouse Cy5 (Dianova) as described elsewhere [25]. A549 cells were harvested with cell dissociation solution (Gibco) and washed twice in PBS. A549 cells ð5 105 Þ or alveolar macrophages ð5 105 Þ were incubated with 100 ng/ml Re F515 LPS at 37 °C in the presence of 0.2% human serum and human recombinant LBP at 0.01, 0.1, 1, and 10 lg=ml for 10 min. Human serum was drawn from healthy volunteers. After washing two times with PBS, cells were incubated with mAb clone 25 followed by an incubation with goat antimouse Cy5 and analyzed by flow cytometry (FACScalibur, Becton Dickinson, Heidelberg, Germany). Human recombinant LBP was expressed in a Baculovirus system as described elsewhere [18]. LPS uptake. A549 cells ð5 105 Þ or alveolar macrophages ð5 105 Þ were harvested and incubated with 100 ng/ml [3 H]LCD25 LPS (List, Biochemicals, CA, USA) in medium containing 0.2% human serum for 10 min at 37 °C in tubes precoated with BSA (0.1%). LBP at 0.01, 0.1, 1.0, and 10 lg=ml or SP-A (generously provided from J.R. Wright, Durham, NC, USA) at 1 and 10 lg=ml) were added to the cells just before LPS was added. If indicated, cells were pre-incubated for 30 min with mAb’s against CD14 (Meml8) and LBP (biG33, Biometec, Greifswald, Germany) at a concentration of 10 lg=ml or cytochalasin D (Sigma–Aldrich, Germany) at a concentration of 0:3 lM. After incubation, cells were washed three times with PBS. Radioactivity was determined in a scintillation counter. All experiments were done at least three times and standard deviations were calculated. Nuclear extraction and electrophoretic mobility shift assay (EMSA). Alveolar macrophages ð4 105 =wellÞ were seeded into 6-well plates in serum-free RPMI (Gibco) and allowed to adhere for 90 min. For stimulation, the medium was replaced by RPMI with 0.2% human serum and 1 ng/ml Re F515 LPS, and indicated amounts of LBP (0.01, 0.1, 1.0, and 10 lg=ml) were added. A549 ð4 105 =wellÞ cells were seeded into 6-well plates in DMEM with 10% FCS and allowed to adhere overnight. Cells were washed two times with PBS and, for stimulation, DMEM with 0.2 % human serum and indicated amounts of LPS, LBP, and IL-1b were added. After incubation for 1 h, nuclear extracts and EMSA were performed as described previously [20]. Experiments were done at least three times and one representative experiment is shown.
Results Materials and methods Cell culture. A549 cells were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) and cultured in Dulbeco’s modified Eagle’s medium (DMEM) (Gibco, Life Technologies GmbH, Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS) (PAA Laboratories,
LBP-dependent activation of alveolar macrophages by LPS is strongly related to LPS uptake Alveolar macrophages are the pivotal lung cells involved in non-specific host defense and are considered to
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play a central role in the regulation of the pulmonary immune response to pathogens and the development of lung inflammation. To investigate dose-dependent effects of LBP on LPS-induced activation of alveolar macrophages, the cells were stimulated with 1 ng/ml Re F515 LPS for 1 h in the presence of increasing amounts of human recombinant LBP and assayed for NF-jB translocation by EMSA. The experiments were done in the presence of 0.2% human serum, a concentration assumed to reflect the physiological serum concentration within the alveolar compartment. We found that NF-jB activation induced by 1 ng/ml LPS is clearly dependent on the presence of LBP with highest activation at 0:1 lg=ml of LBP. Further elevation of the LBP concentration resulted in a strong inhibition of cellular activation (Fig. 1). To determine the effect of various LBP concentrations on LPS binding to the cell surface, we used the same LPS (Re F515) to detect LPS binding by flow cytometry employing the mAb clone 25 against Re F515 LPS. Only weak fluorescence staining was found, however, at LBP concentrations of about 10 lg=ml a slight decrease in LPS binding was observed (Fig. 2A), which corresponded with the decreased activation at the same LBP concentration. The weak fluorescence staining might be due to the high autofluorescences of alveolar macrophages [26] or by rapid internalization of LPS. Professional phagocytes, i.e., alveolar macrophages, are able to internalize 5–70% cell bound LPS, dependent on aggregation state of the LPS, within 1 min [14]. To circumvent these problems, we used radiolabeled Rb LPS from the LCD25 strain of E. coli K12, allowing the detection of the total uptake of LPS which is both cell surface bound and internalized LPS. Incubation of alveolar macrophages with LCD25 LPS in the presence of different amounts of LBP resulted in an enhanced uptake of LPS at 0:1 lg=ml LBP and a strong inhibition of LPS uptake at 10 lg=ml LBP (Fig. 2B). This dose dependency of cellular association of LPS with alveolar macrophages was found to be strongly related to the LPS-induced cellular activation at the corresponding LBP concentration.
Fig. 1. Effects of LBP on LPS-induced NF-jB translocation in rat alveolar macrophages. Alveolar macrophages were stimulated with 1 ng/ml Re F515 LPS and different amounts of LBP at 37 °C in the presence of 0.2% human serum for 1 h and assayed for NFjB translocation by EMSA. Experiments were repeated three times and one representative experiment is shown.
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Fig. 2. Effects of LBP on LPS uptake by alveolar macrophages. (A) Immunostaining of cell surface-bound LPS. Alveolar macrophages were incubated with 100 ng/ml Re F515 LPS at 37 °C in the presence of 0.2% human serum for 10 min. Increasing amounts of LBP were added as indicated. Surface-bound LPS was measured by immunofluorescence staining using mAb (A25) against Re F515 LPS and goat anti-mouse secondary mAb. (B) LPS uptake. Alveolar macrophages were incubated with 10 ng/ml tritium-labeled LCD25 LPS at 37 °C in the presence of 0.2% human serum for 10 min. Increasing amounts of LBP were added as indicated. LPS uptake was determined as counts per minute per 500.000 cells. Experiments were repeated three times. Mean and SD are indicated. * p < 0:05 compared to LPS uptake in the absence of LBP.
High concentrations of LBP mediate an increased LPS uptake by A549 cells but do not result in cellular activation Beside alveolar macrophages respiratory type II cells are an integral component of the pulmonary
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innate immunity secreting immunmodulatory molecules such as surfactant proteins, IL-8 and others [27]. Recently, respiratory type II cells have been shown to produce LPB upon proinflammatory stimulation in vitro, possibly explaining elevated LBP levels in the inflamed lung [28]. Therefore, we investigated the effects of increasing concentrations of LBP on LPSbinding and uptake by the respective lung epithelial cell line A549. Employing Re F515 LPS, we found a LBP-dependent binding of LPS to A549 cells detected by flow cytometry. LBP at a concentration of 0:1 lg=ml is sufficient to elicit maximal LPS binding. Further increase of the LBP concentration has no additional effect on LPS binding (Fig. 3A). Using radiolabeled LCD 25 LPS we got slightly different results. In contrast to Re F515 LPS, uptake of LCD25 LPS was further elevated by increasing the LBP concentration from 0:1 lg=ml to finally 10 lg=ml. This difference may be due to the fact that both cell surface bound and internalized LPS were determined. Pulmonary surfactant protein A (SP-A), an innate immune molecule that binds to R-LPS and enhances R-LPS uptake by alveolar macrophages in a dose- and time-dependent manner [29] served as a control for LBP effects. In contrast to LBP, SP-A did not enhance LPS uptake by A549 cells, confirming that the effect of LBP is specific. The SP-A preparation used in the current experiments also increased LPS uptake by alveolar macrophages (data not shown). To test whether the enhanced LPS uptake by A549 cells at high concentrations of LBP is accompanied by cellular activation, we performed NF-jB gelshift analysis. As shown in Fig. 4A, LPS does not activate NF-jB translocation in A549 cells even at a high concentration of LBP. In contrast, stimulation with IL-1b, known to induce LBP release from these cells [21], resulted in a strong NF-jB translocation showing that downstream signaling molecules of the IL-1R/TLR pathway are functional (Fig. 4A).
LBP-mediated enhancement of LPS uptake by A549 cells partially depends on mCD14 and LPS internalization To test whether the LPS receptors, mCD14 and TLR4, responsible for LPS-binding and signaling are expressed on A549 cells, we performed flow cytometry analysis. As shown in Fig. 5, we found that A549 cells do express mCD14 but no TLR-4. Next we tested whether the effect of LBP on LPS uptake by A549 cells is mCD14-dependent and whether LPS internalization by these cells is involved. Preincubation of the cells with the monoclonal anti-LBP Ab biG33 or with monoclonal anti-CD14 Ab Mem18 resulted in an inhibition of LBPmediated LPS-uptake (Fig. 4B). Pretreatment of the cells with biG33 resulted in an 65% inhibition of LPS
Fig. 3. Effects of LBP on LPS uptake by A549 cells. (A) Immunostaining of cell surface-bound LPS. A549 cells were incubated with 100 ng/ml Re F515 LPS at 37 °C in the presence of 0.2% human serum for 10 min. Increasing amounts of LBP were added as indicated. Surface-bound LPS was measured by immunofluorescence staining using mAb (A25) against Re F515 LPS and goat anti-mouse secondary mAb. (B) LPS uptake. A549 cells were incubated with 100 ng/ml tritium-labeled LCD25 LPS at 37 °C in the presence of 0.2% human serum for 10 min. Increasing amounts of LBP were added as indicated. LPS uptake was determined as counts per minute per 500.000 cells. All experiments were repeated three times. Mean and SD are indicated. *p < 0:05, and **p < 0:01 compared to LPS uptake in the absence of LBP.
uptake. Taking into account that LPS uptake without the addition of LBP is reduced to 35% compared to LPS uptake in the presence of 1 lg=ml LBP (Fig. 3B) addition of biG33 resulted in a total inhibition of LBP mediated LPS uptake. Blocking mCD14 with Mem18 resulted in only 40% inhibition of LPS uptake. The addition of 0:3 lM cytochalasin D to the medium resulted in 60% decrease of LPS uptake indicating that the LBP-mediated LPS uptake is nearly completely dependent on internalization.
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Fig. 5. Expression of mCD14 and TLR-4 by A549 cells. Cells were analyzed for the expression of mCD14 and TLR-4 by flow cytometry using mAb’s Mem18 against CD14 and HAT 125 against human TLR-4. Respective isotype matched mAb’s were used as control.
Fig. 4. (A) Effects of LPS, LPS plus LBP, or IL-1b on NF-jB translocation in A549 cells. A549 cells were incubated with Re F515 LPS (100 ng/ml), Re F515 LPS (100 ng/ml) plus LBP (1 lg=ml LBP), or IL-1b (1 ng/ml) at 37 °C in the presence of 0.2% human serum. After 1 h of incubation, cells were assayed for NF-jB translocation by EMSA. Experiments were repeated three times and one representative experiment is shown. (B) Effects of anti-CD 14, anti-LBP, and cytochalsin D pretreatment of A549 cells on LBP-mediated LPS uptake. A549 cells were pretreated with mAb biG33 against LBP ð10 lg=mlÞ, Mem18 against CD14 ð10 lg=mlÞ, or cytochalasin D ð0:3 lMÞ for 30 min at 37 °C. After preincubation, the cells were incubated with 100 ng/ml tritium-labeled LCD25 LPS at 37 °C in the presence of 0.2% human serum and 1 lg=ml LBP for 10 min. LPS uptake was determined as counts per minute per 500.000 cells. The inhibition of LPS uptake is expressed as percentage of control (no preincubation with anti-LBP, anti-CD 14, or cytochalasin D). Experiments were repeated three times. Mean and SD are indicated. **p < 0:01 compared to LPS uptake in the absence of anti-LBP, anti-CD14, or cytochalasin D.
Discussion Whereas numerous studies emphasize the need for a regulated release of cytokines upon the recognition of LPS to combat Gram-negative infections [11,30] the dysregulated release of proinflammatory mediators
induced by LPS is the key event in developing Gramnegative sepsis or septic shock. Cellular recognition of and cellular activation by LPS are strongly regulated by LBP. For cells to sense low amounts of LPS to mount a immune reaction against Gram-negative bacteria, LBP is absolutely necessary [11,30]. On the other hand, LBP has detrimental effects in models of endotoxemia since monoclonal antibodies to LBP have been shown to rescue mice from LPS-induced shock [32,33]. Involvement of LBP in LPS clearance by HDL reflects also the oppositional effects of LBP on LPS-induced activation of innate immunity [15,34]. We have shown previously that high concentrations of LBP, similar to that detected in acute phase situations, have beneficial effects in a mouse model of bacteriemia and sepsis [18]. In addition, we previously reported that high concentrations of LBP can inhibit LPS-induced TNF-a production by human monocytes [19]. Since local expression of LBP by lung epithelial cells upon proinflammatory stimulation has been documented [21], we investigated whether enhanced concentrations of LBP may influence LPS-induced cellular activation of lung specific immunocompetent cells, which are mainly constituted by alveolar macrophages and epithelial cells. We first analyzed the effect of LBP on LPS-induced activation of alveolar macrophages and found that increased amounts of LBP up to 100 ng/ml result in an enhanced activation as determined by NF-jB translocation. This finding corroborates data published by Martin and co-workers [23] who found that increasing amounts of LBP up to 100 ng/ml result in an increased TNF-a production by
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alveolar macrophages. In contrast, higher concentrations of LBP, comparable to LBP concentraions in alveolar fluids found in patients with bacterial pneumonia, resulted in a reduced NF-jB translocation. LPS uptake under the similar experimental conditions was strongly related to LPS-induced cellular activation. This correlation of LPS-induced activation and LPS uptake was also shown for human monocytes. In that study we have shown that the inhibitory effect of severe sepsis sera on monocyte activation is dependent on elevated LBP concentrations during acute phase situations. Furthermore, elevated LBP concentrations in severe sepsis sera were also responsible for decreased LPS uptake by human monocytes in the presence of sepsis sera [19]. Lung epithelial cells, such as respiratory type II cells, are important immunocompetent cells which are able to produce immune-modulatory proteins, among them surfactant proteins and IL-8. Furthermore, these cells as well as the respective cell line A549 are able to express LBP upon proinflammatory stimulation [21]. Therefore, we measured the effect of high concentrations of LBP on LPS uptake and LPS-induced signaling by A549 cells. We found that increased levels of LBP result in enhanced LPS uptake by A549 cells which can be completely inhibited by the addition of a mAb against LBP. This LBP-mediated LPS uptake was found to partly depend on mCD14 and LPS internalization as shown by blocking mCD14 with a mAb and blocking internalization by preincubation with cytochalasin D. The expression of mCD14 by A549 cells is very low compared to human monocytes (data not shown) and reported here for the first time. However, human umbelical vein cells (HUVEC), an endothelial cell line that has been thought to be mCD14 negative was finally shown to express low amounts of mCD14 [35]. In vivo, extramyeloid expression of the murine CD14 gene has been documented [36]. Importantly, these investigators found that the lung epithelium does express mCD14 both constitutively and inducible upon challenge with LPS [36]. LPS binding to mCD14 and subsequent internalization is a prerequisite for LPS clearance and detoxification [37] that has been attributed to cells of the myeloid lineage due to the paradigm of restricted mCD14 expression. Low amounts of mCD14 expression on epithelial or endothelial cells, as shown in the current report and others, may enable cells of epithelial origin to participate in LPS internalization and detoxification. Until now, the responsiveness of respiratory type II- and A549 cells is not definitely cleared. There are some reports showing that A549 cells are not responsive for LPS until they have been transfected with the adenoviral E1A gene [38,39]. Other investigators found a LPS-induced IL-8 secretion by A549 cells [40,41]. In these studies very high concentrations of LPS were used. Specially, Koyama and
co-workers used up to 100 lg=ml LPS to induce IL-8 secretion by A549 cells, furthermore, they found highest activity using a LPS preparation from Pseudomonas aeruginosa whereas various E. coli LPS’s showed a 10-fold lower activity [40]. However, in our hands A549 cells cannot be stimulated by LPS to translocate NF-jB even at a concentration of LBP that mediate enhanced uptake of LPS. The lack of TLR-4 expression should explain the unresponsiveness of A549 cells towards LPS. The mechanisms responsible for the differences in LPS uptake byalveolar macrophages and A549 cells at high LBP concentrations remain to be investigated. In summary, we propose that an enhanced expression of LBP by respiratory type II cells upon proinflammatory stimulation may have protective effects within the lungs via inhibiting LPS-induced activation of alveolar macrophages and/or via increasing a ‘‘silent’’ uptake of LPS by lung epithelial cells. This ‘‘silent’’ LPS uptake may be a general mechanism by which LPS unresponsive cells that express low amounts of mCD14 may participate in LPS clearance.
Acknowledgments We thank V. Horesji (Institute for Molecular Genetics, Prague, Czech Republic) for providing anti-CD 14 mAb, clone MEM18, and Katrin Klopfenstein and Andrea Sager for their excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 367, Projekt C5; DFG, SCH828/1-6).
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Inhibition of LPS-induced activation of alveolar macrophages by high concentrations of LPS-binding protein Lutz Hamann,a,b,* Cordula Stamme,c,d Artur J. Ulmer,a and Ralf R. Schumannb a Department of Immunology and Cell Biology, Center for Medicine and Bioscience, Borstel, Germany Institute of Microbiology and Hygiene, Charit e, Humboldt University Berlin, Dorotheenstraße 96, 10117 Berlin, Germany Department of Immunochemistry and Biochemical Microbiology Research Center Borstel, Center for Medicine and Bioscience, Borstel, Germany d Department of Anesthesiology, University of L€ubeck, L€ubeck, Germany b
c
Received 10 June 2002
Abstract Lipopolysaccharide (LPS)-binding protein regulates the effects of LPS on immunocompetent cells. By catalyzing the binding of LPS to membrane CD14, LPS-binding protein (LBP) potentiates both the inflammatory response and internalization of LPS. LBPmediated transport of LPS into high density lipoprotein particles participates in LPS clearance. Elevated serum levels of LBP have been shown to elicit protective effects in vivo. Because the expression of LBP is upregulated in lung epithelial cells upon proinflammatory stimulation, we here investigated whether LBP modulates inflammatory responses by lung specific cells. The moderate elevation of LBP concentrations enhanced both LPS-induced signaling and LPS uptake by rat alveolar macrophages, whereas strongly elevated LBP levels inhibited both. In contrast, the lung epithelial cell line A549 responded to high concentrations of LBP by an enhanced LPS uptake which did not result in cellular activation, suggesting an anti-inflammatory function of these cells by clearing LPS. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Inflammation; LPS-induced signaling; LPS uptake; Innate immunity; Alveolar macrophages; Lung epithelial
Lipopolysaccaride (LPS), the major cell wall component of Gram-negative bacteria, is a strong activator of the innate immune system triggering the release of various proinflammatory mediators, among them cytokines such as TNF-a, IL-1b, and IL-6 [1–3]. The effects of LPS on immunocompetent cells are strongly regulated by LPSbinding protein (LBP) in different ways. The most prominent feature of LBP is to catalyze the binding of LPS to the LPS receptor mCD14 by releasing LPS monomers from LPS micells and transfering them to mCD14 [4–6]. LPS binding to mCD14, mainly expressed on monocytes and macrophages, initiates a signaling cascade via toll-like receptor 4 and MD-2 [7,8]. Immune cells that do not express mCD14, such as endothelial cells and smooth muscle cells, can be stimulated by LPS to release cytokines via soluble CD14 (sCD14) [9]. The crucial role of LBP in recognizing LPS as well as whole bacteria has been shown by employing LBP knock out *
Corresponding author. Fax: +49-30-45052-24904. E-mail address: [email protected] (L. Hamann).
mice. Blood cells from LBP)/) mice do not release TNFa in response to LPS [10]. In vivo, LBP)/) mice fail to combat a Gram-negative infection with Salmonella typhimurium [11]. However, a dysbalanced release of these mediators triggers the onset of sepsis or septic shock [12]. Binding to mCD14 also results in LPS internalization and subsequent detoxification of LPS by professional phagocytes [13,14]. The internalization of LPS has been attributed to cells of the myeloid lineage, mainly monocytes and macrophages, due to the restricted expression pattern of mCD14 by these cells. On the other hand, LBP is associated with high density lipoproteins (HDL) particles and LPS-binding to LBP can result in LPS clearance via HDL particles [15]. In acute phase situations, the LBP expression by hepatocytes, which are considered to be the main source of LBP [16], is strongly upregulated by proinflammatory cytokines reaching serum levels of about 200 lg=ml [17]. We have recently shown that these concentrations of LBP have protective effects in a mouse model of bacteriemia and sepsis [18] and that high LBP concentrations can
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inhibit LPS induced TNF-a production by human monocytes [19]. Furthermore, we have shown that enhanced LBP levels mediate enhanced LPS uptake by Chinese hamster ovary cells without cellular activation which may be a mechanism for LPS clearance [20]. Recently, Dentener and colleagues [21] have documented that human respiratory type II cells are able to express LBP in response to IL-1b and IL-6 both of which might be released in vivo by LPS-stimulated alveolar macrophages. LBP concentrations of 100 ng/ml have been detected in broncho alveolar lavage (BAL) of patients with bacterial pneumonia [22]. Assuming that BAL dilutes alveolar fluids by 50–100-fold LBP concentrations should be much higher. The function of this elevated LBP expression is not known. Martin and coworkers [23] have shown in vitro that an increase of LBP concentration up to 100 ng/ml results in an enhanced activation of alveolar macrophages by LPS. However, due to the fact that the local expression of LBP by the lung epithelium is inducible by proinflammatory cytokines, LBP concentrations may reach significantly higher levels in the pulmonary compartment under proinflammatory conditions. It might be assumed that this locally increased LBP production results in a similar inhibition of LPS-induced signaling as it has been shown for elevated LBP concentrations employing a mouse model of sepsis and bacteriemia [18]. In the current study, we show that the effect of LBP on LPS uptake and LPS-induced signaling significantly varies with both the cell types, i.e., alveolar macrophages or type II cells, and the LBP concentration used. In rat alveolar macrophages, LBP concentrations up to 100 ng/ ml resulted in a dose-dependent increase of both LPS uptake and LPS-induced signaling. Further increase of the LBP concentration resulted in a remarkable inhibition of LPS-induced signaling and LPS uptake by these cells. In contrast, A549 cells, a respiratory type II cell line, showed an increase in LPS uptake by increasing LBP concentrations. The LBP-mediated increase of LPS uptake by A549 cells did not result in cellular activation, was found to be significantly inhibited by cytochalasin D, and was partially dependent on mCD14. Therefore, LBP concentrations higher than 100 ng/ml may result in the inhibition of LPS-induced signaling in vivo by either of two mechanisms. First, decrease of cellular association of LPS associated with an inhibited LPS-induced signaling by alveolar macrophages. Second, increase of an competitive LPS uptake by LPS unresponsive cells which may be a new mechanism of LPS clearance.
Linz, Austria), penicillin (100 U/ml), and streptomycin ð100 lg=mlÞ. The cells were grown at 37 °C in an atmosphere containing 5% CO2 . Alveolar macrophages were isolated by lung lavage of specific pathogen-free, male Sprague–Dawley rat weighing 250–300 g. Rats were anesthetized with pentobarbital and killed by exsanguination. The lungs were removed and lavaged six times with a macrophage isolation buffer containing 140 mM NaCl, 6 mM glucose, 2.5 mM phosphate buffer, 10 mM HEPES, and 0.2 mM EGTA, followed by two lavages with the same buffer containing 1.3 mM magnesium and 2 mM calcium and no EGTA. Samples were centrifuged at 200g for 10 min. Cell recovery routinely averaged 5–7 106 cells/animal. The viability of the cells was determined by erythrosin B exclusion and averaged 95–98%. Flow cytometry. Cell surface expression of mCD14 and TLR-4 was determined by flow cytometry using mAb’s Mem18 against CD14 (kindly provided by V. Horesji, Prague, Cezch Republic), HAT 125 against TLR-4 (kindly provided by K. Myake, Saga, Japan) and goat anti-mouse FITC as secondary antibody (Dianova, Hamburg, Germany). Cell surface binding of Re F515 LPS from the mutant Escherichia coli strain F515 (kind gift of H. Brade, Borstel, Germany) was assessed by immunofluorescence employing the anti-Re F515 LPS mAb clone A25 [24], and goat anti-mouse Cy5 (Dianova) as described elsewhere [25]. A549 cells were harvested with cell dissociation solution (Gibco) and washed twice in PBS. A549 cells ð5 105 Þ or alveolar macrophages ð5 105 Þ were incubated with 100 ng/ml Re F515 LPS at 37 °C in the presence of 0.2% human serum and human recombinant LBP at 0.01, 0.1, 1, and 10 lg=ml for 10 min. Human serum was drawn from healthy volunteers. After washing two times with PBS, cells were incubated with mAb clone 25 followed by an incubation with goat antimouse Cy5 and analyzed by flow cytometry (FACScalibur, Becton Dickinson, Heidelberg, Germany). Human recombinant LBP was expressed in a Baculovirus system as described elsewhere [18]. LPS uptake. A549 cells ð5 105 Þ or alveolar macrophages ð5 105 Þ were harvested and incubated with 100 ng/ml [3 H]LCD25 LPS (List, Biochemicals, CA, USA) in medium containing 0.2% human serum for 10 min at 37 °C in tubes precoated with BSA (0.1%). LBP at 0.01, 0.1, 1.0, and 10 lg=ml or SP-A (generously provided from J.R. Wright, Durham, NC, USA) at 1 and 10 lg=ml) were added to the cells just before LPS was added. If indicated, cells were pre-incubated for 30 min with mAb’s against CD14 (Meml8) and LBP (biG33, Biometec, Greifswald, Germany) at a concentration of 10 lg=ml or cytochalasin D (Sigma–Aldrich, Germany) at a concentration of 0:3 lM. After incubation, cells were washed three times with PBS. Radioactivity was determined in a scintillation counter. All experiments were done at least three times and standard deviations were calculated. Nuclear extraction and electrophoretic mobility shift assay (EMSA). Alveolar macrophages ð4 105 =wellÞ were seeded into 6-well plates in serum-free RPMI (Gibco) and allowed to adhere for 90 min. For stimulation, the medium was replaced by RPMI with 0.2% human serum and 1 ng/ml Re F515 LPS, and indicated amounts of LBP (0.01, 0.1, 1.0, and 10 lg=ml) were added. A549 ð4 105 =wellÞ cells were seeded into 6-well plates in DMEM with 10% FCS and allowed to adhere overnight. Cells were washed two times with PBS and, for stimulation, DMEM with 0.2 % human serum and indicated amounts of LPS, LBP, and IL-1b were added. After incubation for 1 h, nuclear extracts and EMSA were performed as described previously [20]. Experiments were done at least three times and one representative experiment is shown.
Results Materials and methods Cell culture. A549 cells were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) and cultured in Dulbeco’s modified Eagle’s medium (DMEM) (Gibco, Life Technologies GmbH, Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS) (PAA Laboratories,
LBP-dependent activation of alveolar macrophages by LPS is strongly related to LPS uptake Alveolar macrophages are the pivotal lung cells involved in non-specific host defense and are considered to
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play a central role in the regulation of the pulmonary immune response to pathogens and the development of lung inflammation. To investigate dose-dependent effects of LBP on LPS-induced activation of alveolar macrophages, the cells were stimulated with 1 ng/ml Re F515 LPS for 1 h in the presence of increasing amounts of human recombinant LBP and assayed for NF-jB translocation by EMSA. The experiments were done in the presence of 0.2% human serum, a concentration assumed to reflect the physiological serum concentration within the alveolar compartment. We found that NF-jB activation induced by 1 ng/ml LPS is clearly dependent on the presence of LBP with highest activation at 0:1 lg=ml of LBP. Further elevation of the LBP concentration resulted in a strong inhibition of cellular activation (Fig. 1). To determine the effect of various LBP concentrations on LPS binding to the cell surface, we used the same LPS (Re F515) to detect LPS binding by flow cytometry employing the mAb clone 25 against Re F515 LPS. Only weak fluorescence staining was found, however, at LBP concentrations of about 10 lg=ml a slight decrease in LPS binding was observed (Fig. 2A), which corresponded with the decreased activation at the same LBP concentration. The weak fluorescence staining might be due to the high autofluorescences of alveolar macrophages [26] or by rapid internalization of LPS. Professional phagocytes, i.e., alveolar macrophages, are able to internalize 5–70% cell bound LPS, dependent on aggregation state of the LPS, within 1 min [14]. To circumvent these problems, we used radiolabeled Rb LPS from the LCD25 strain of E. coli K12, allowing the detection of the total uptake of LPS which is both cell surface bound and internalized LPS. Incubation of alveolar macrophages with LCD25 LPS in the presence of different amounts of LBP resulted in an enhanced uptake of LPS at 0:1 lg=ml LBP and a strong inhibition of LPS uptake at 10 lg=ml LBP (Fig. 2B). This dose dependency of cellular association of LPS with alveolar macrophages was found to be strongly related to the LPS-induced cellular activation at the corresponding LBP concentration.
Fig. 1. Effects of LBP on LPS-induced NF-jB translocation in rat alveolar macrophages. Alveolar macrophages were stimulated with 1 ng/ml Re F515 LPS and different amounts of LBP at 37 °C in the presence of 0.2% human serum for 1 h and assayed for NFjB translocation by EMSA. Experiments were repeated three times and one representative experiment is shown.
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Fig. 2. Effects of LBP on LPS uptake by alveolar macrophages. (A) Immunostaining of cell surface-bound LPS. Alveolar macrophages were incubated with 100 ng/ml Re F515 LPS at 37 °C in the presence of 0.2% human serum for 10 min. Increasing amounts of LBP were added as indicated. Surface-bound LPS was measured by immunofluorescence staining using mAb (A25) against Re F515 LPS and goat anti-mouse secondary mAb. (B) LPS uptake. Alveolar macrophages were incubated with 10 ng/ml tritium-labeled LCD25 LPS at 37 °C in the presence of 0.2% human serum for 10 min. Increasing amounts of LBP were added as indicated. LPS uptake was determined as counts per minute per 500.000 cells. Experiments were repeated three times. Mean and SD are indicated. * p < 0:05 compared to LPS uptake in the absence of LBP.
High concentrations of LBP mediate an increased LPS uptake by A549 cells but do not result in cellular activation Beside alveolar macrophages respiratory type II cells are an integral component of the pulmonary
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innate immunity secreting immunmodulatory molecules such as surfactant proteins, IL-8 and others [27]. Recently, respiratory type II cells have been shown to produce LPB upon proinflammatory stimulation in vitro, possibly explaining elevated LBP levels in the inflamed lung [28]. Therefore, we investigated the effects of increasing concentrations of LBP on LPSbinding and uptake by the respective lung epithelial cell line A549. Employing Re F515 LPS, we found a LBP-dependent binding of LPS to A549 cells detected by flow cytometry. LBP at a concentration of 0:1 lg=ml is sufficient to elicit maximal LPS binding. Further increase of the LBP concentration has no additional effect on LPS binding (Fig. 3A). Using radiolabeled LCD 25 LPS we got slightly different results. In contrast to Re F515 LPS, uptake of LCD25 LPS was further elevated by increasing the LBP concentration from 0:1 lg=ml to finally 10 lg=ml. This difference may be due to the fact that both cell surface bound and internalized LPS were determined. Pulmonary surfactant protein A (SP-A), an innate immune molecule that binds to R-LPS and enhances R-LPS uptake by alveolar macrophages in a dose- and time-dependent manner [29] served as a control for LBP effects. In contrast to LBP, SP-A did not enhance LPS uptake by A549 cells, confirming that the effect of LBP is specific. The SP-A preparation used in the current experiments also increased LPS uptake by alveolar macrophages (data not shown). To test whether the enhanced LPS uptake by A549 cells at high concentrations of LBP is accompanied by cellular activation, we performed NF-jB gelshift analysis. As shown in Fig. 4A, LPS does not activate NF-jB translocation in A549 cells even at a high concentration of LBP. In contrast, stimulation with IL-1b, known to induce LBP release from these cells [21], resulted in a strong NF-jB translocation showing that downstream signaling molecules of the IL-1R/TLR pathway are functional (Fig. 4A).
LBP-mediated enhancement of LPS uptake by A549 cells partially depends on mCD14 and LPS internalization To test whether the LPS receptors, mCD14 and TLR4, responsible for LPS-binding and signaling are expressed on A549 cells, we performed flow cytometry analysis. As shown in Fig. 5, we found that A549 cells do express mCD14 but no TLR-4. Next we tested whether the effect of LBP on LPS uptake by A549 cells is mCD14-dependent and whether LPS internalization by these cells is involved. Preincubation of the cells with the monoclonal anti-LBP Ab biG33 or with monoclonal anti-CD14 Ab Mem18 resulted in an inhibition of LBPmediated LPS-uptake (Fig. 4B). Pretreatment of the cells with biG33 resulted in an 65% inhibition of LPS
Fig. 3. Effects of LBP on LPS uptake by A549 cells. (A) Immunostaining of cell surface-bound LPS. A549 cells were incubated with 100 ng/ml Re F515 LPS at 37 °C in the presence of 0.2% human serum for 10 min. Increasing amounts of LBP were added as indicated. Surface-bound LPS was measured by immunofluorescence staining using mAb (A25) against Re F515 LPS and goat anti-mouse secondary mAb. (B) LPS uptake. A549 cells were incubated with 100 ng/ml tritium-labeled LCD25 LPS at 37 °C in the presence of 0.2% human serum for 10 min. Increasing amounts of LBP were added as indicated. LPS uptake was determined as counts per minute per 500.000 cells. All experiments were repeated three times. Mean and SD are indicated. *p < 0:05, and **p < 0:01 compared to LPS uptake in the absence of LBP.
uptake. Taking into account that LPS uptake without the addition of LBP is reduced to 35% compared to LPS uptake in the presence of 1 lg=ml LBP (Fig. 3B) addition of biG33 resulted in a total inhibition of LBP mediated LPS uptake. Blocking mCD14 with Mem18 resulted in only 40% inhibition of LPS uptake. The addition of 0:3 lM cytochalasin D to the medium resulted in 60% decrease of LPS uptake indicating that the LBP-mediated LPS uptake is nearly completely dependent on internalization.
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Fig. 5. Expression of mCD14 and TLR-4 by A549 cells. Cells were analyzed for the expression of mCD14 and TLR-4 by flow cytometry using mAb’s Mem18 against CD14 and HAT 125 against human TLR-4. Respective isotype matched mAb’s were used as control.
Fig. 4. (A) Effects of LPS, LPS plus LBP, or IL-1b on NF-jB translocation in A549 cells. A549 cells were incubated with Re F515 LPS (100 ng/ml), Re F515 LPS (100 ng/ml) plus LBP (1 lg=ml LBP), or IL-1b (1 ng/ml) at 37 °C in the presence of 0.2% human serum. After 1 h of incubation, cells were assayed for NF-jB translocation by EMSA. Experiments were repeated three times and one representative experiment is shown. (B) Effects of anti-CD 14, anti-LBP, and cytochalsin D pretreatment of A549 cells on LBP-mediated LPS uptake. A549 cells were pretreated with mAb biG33 against LBP ð10 lg=mlÞ, Mem18 against CD14 ð10 lg=mlÞ, or cytochalasin D ð0:3 lMÞ for 30 min at 37 °C. After preincubation, the cells were incubated with 100 ng/ml tritium-labeled LCD25 LPS at 37 °C in the presence of 0.2% human serum and 1 lg=ml LBP for 10 min. LPS uptake was determined as counts per minute per 500.000 cells. The inhibition of LPS uptake is expressed as percentage of control (no preincubation with anti-LBP, anti-CD 14, or cytochalasin D). Experiments were repeated three times. Mean and SD are indicated. **p < 0:01 compared to LPS uptake in the absence of anti-LBP, anti-CD14, or cytochalasin D.
Discussion Whereas numerous studies emphasize the need for a regulated release of cytokines upon the recognition of LPS to combat Gram-negative infections [11,30] the dysregulated release of proinflammatory mediators
induced by LPS is the key event in developing Gramnegative sepsis or septic shock. Cellular recognition of and cellular activation by LPS are strongly regulated by LBP. For cells to sense low amounts of LPS to mount a immune reaction against Gram-negative bacteria, LBP is absolutely necessary [11,30]. On the other hand, LBP has detrimental effects in models of endotoxemia since monoclonal antibodies to LBP have been shown to rescue mice from LPS-induced shock [32,33]. Involvement of LBP in LPS clearance by HDL reflects also the oppositional effects of LBP on LPS-induced activation of innate immunity [15,34]. We have shown previously that high concentrations of LBP, similar to that detected in acute phase situations, have beneficial effects in a mouse model of bacteriemia and sepsis [18]. In addition, we previously reported that high concentrations of LBP can inhibit LPS-induced TNF-a production by human monocytes [19]. Since local expression of LBP by lung epithelial cells upon proinflammatory stimulation has been documented [21], we investigated whether enhanced concentrations of LBP may influence LPS-induced cellular activation of lung specific immunocompetent cells, which are mainly constituted by alveolar macrophages and epithelial cells. We first analyzed the effect of LBP on LPS-induced activation of alveolar macrophages and found that increased amounts of LBP up to 100 ng/ml result in an enhanced activation as determined by NF-jB translocation. This finding corroborates data published by Martin and co-workers [23] who found that increasing amounts of LBP up to 100 ng/ml result in an increased TNF-a production by
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alveolar macrophages. In contrast, higher concentrations of LBP, comparable to LBP concentraions in alveolar fluids found in patients with bacterial pneumonia, resulted in a reduced NF-jB translocation. LPS uptake under the similar experimental conditions was strongly related to LPS-induced cellular activation. This correlation of LPS-induced activation and LPS uptake was also shown for human monocytes. In that study we have shown that the inhibitory effect of severe sepsis sera on monocyte activation is dependent on elevated LBP concentrations during acute phase situations. Furthermore, elevated LBP concentrations in severe sepsis sera were also responsible for decreased LPS uptake by human monocytes in the presence of sepsis sera [19]. Lung epithelial cells, such as respiratory type II cells, are important immunocompetent cells which are able to produce immune-modulatory proteins, among them surfactant proteins and IL-8. Furthermore, these cells as well as the respective cell line A549 are able to express LBP upon proinflammatory stimulation [21]. Therefore, we measured the effect of high concentrations of LBP on LPS uptake and LPS-induced signaling by A549 cells. We found that increased levels of LBP result in enhanced LPS uptake by A549 cells which can be completely inhibited by the addition of a mAb against LBP. This LBP-mediated LPS uptake was found to partly depend on mCD14 and LPS internalization as shown by blocking mCD14 with a mAb and blocking internalization by preincubation with cytochalasin D. The expression of mCD14 by A549 cells is very low compared to human monocytes (data not shown) and reported here for the first time. However, human umbelical vein cells (HUVEC), an endothelial cell line that has been thought to be mCD14 negative was finally shown to express low amounts of mCD14 [35]. In vivo, extramyeloid expression of the murine CD14 gene has been documented [36]. Importantly, these investigators found that the lung epithelium does express mCD14 both constitutively and inducible upon challenge with LPS [36]. LPS binding to mCD14 and subsequent internalization is a prerequisite for LPS clearance and detoxification [37] that has been attributed to cells of the myeloid lineage due to the paradigm of restricted mCD14 expression. Low amounts of mCD14 expression on epithelial or endothelial cells, as shown in the current report and others, may enable cells of epithelial origin to participate in LPS internalization and detoxification. Until now, the responsiveness of respiratory type II- and A549 cells is not definitely cleared. There are some reports showing that A549 cells are not responsive for LPS until they have been transfected with the adenoviral E1A gene [38,39]. Other investigators found a LPS-induced IL-8 secretion by A549 cells [40,41]. In these studies very high concentrations of LPS were used. Specially, Koyama and
co-workers used up to 100 lg=ml LPS to induce IL-8 secretion by A549 cells, furthermore, they found highest activity using a LPS preparation from Pseudomonas aeruginosa whereas various E. coli LPS’s showed a 10-fold lower activity [40]. However, in our hands A549 cells cannot be stimulated by LPS to translocate NF-jB even at a concentration of LBP that mediate enhanced uptake of LPS. The lack of TLR-4 expression should explain the unresponsiveness of A549 cells towards LPS. The mechanisms responsible for the differences in LPS uptake byalveolar macrophages and A549 cells at high LBP concentrations remain to be investigated. In summary, we propose that an enhanced expression of LBP by respiratory type II cells upon proinflammatory stimulation may have protective effects within the lungs via inhibiting LPS-induced activation of alveolar macrophages and/or via increasing a ‘‘silent’’ uptake of LPS by lung epithelial cells. This ‘‘silent’’ LPS uptake may be a general mechanism by which LPS unresponsive cells that express low amounts of mCD14 may participate in LPS clearance.
Acknowledgments We thank V. Horesji (Institute for Molecular Genetics, Prague, Czech Republic) for providing anti-CD 14 mAb, clone MEM18, and Katrin Klopfenstein and Andrea Sager for their excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 367, Projekt C5; DFG, SCH828/1-6).
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