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Original Research |

Effects of Telithromycin in In Vitro and In Vivo Models of Lipopolysaccharide-Induced Airway Inflammation* FREE TO VIEW

Magdalena Leiva, PhD; Alfonso Ruiz-Bravo, PhD; Maria Jimenez-Valera, PhD
Author and Funding Information

*From the Microbial Immunology Research Group, Department of Microbiology, Faculty of Pharmacy, University of Granada, Granada, Spain.

Correspondence to: Maria Jimenez-Valera, PhD, Department of Microbiology, Faculty of Pharmacy, University of Granada, Granada 18071, Spain; e-mail: aruizbr@ugr.es


Chest. 2008;134(1):20-29. doi:10.1378/chest.07-3056
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Background: The ketolide antibiotic telithromycin (TEL) exerts immunomodulatory and antiinflammatory effects in vitro and in a mouse model of septic shock. We studied the antiinflammatory activity of TEL in in vitro and in vivo models of airway inflammation induced by lipopolysaccharide (LPS).

Methods: We measured the effects of TEL on the response of RAW 264.7 macrophages to LPS and of murine lung epithelial (MLE)-12 cells to supernatants of LPS-stimulated RAW 264.7 macrophages. Macrophage inflammatory protein (MIP)-2 and tumor necrosis factor (TNF)-α production, nuclear factor (NF)-κB activation, and apoptosis were determined. Acute airway inflammation was induced in untreated and TEL-treated BALB/c mice by nebulization with LPS. Total number of leukocytes, macrophages, and neutrophils, the protein concentration, and nitrite and cytokine levels were determined in the BAL fluid.

Results: TEL inhibited in a dose-dependent manner the production of MIP-2 and TNF-α by LPS-stimulated RAW 264.7 macrophages, and the production of MIP-2 by MLE-12 epithelial cells to supernatants of LPS-stimulated RAW 264.7 macrophages. NF-κB activation was inhibited and apoptosis was increased in both cell lines by TEL. The LPS-induced influx of neutrophils in BAL fluid was decreased by TEL pretreatment. TEL also reduced protein, nitrite, MIP-2, and TNF-α levels in the BAL fluid of LPS-nebulized animals.

Conclusions: We have provided evidence that TEL exerts potent antiinflammatory effects in LPS-induced airways injury. We propose that TEL acts in the early phase of inflammation by reducing the release of inflammatory mediators through NF-κB inhibition, and in the later phase through enhancement of inflammatory cell apoptosis.

Figures in this Article

Airway inflammation is a common feature of infectious pulmonary diseases. Although the inflammatory response clearly contributes to the reduction of the number of pathogens obtained from the site of infection, a prolonged inflammatory response might worsen lung injury. Alveolar macrophages are the first line of innate cell-mediated immunity in the lower respiratory tract.12 The lipopolysaccharide (LPS) produced by Gram-negative bacteria is an important inducer of lung injury. Through the activation of the transcription factor nuclear factor (NF)-κB, LPS causes the release by macrophages of inflammatory mediators such as tumor necrosis factor (TNF)-α and chemokines, and the expression of inducible nitric oxide synthase (iNOS)35 Although alveolar macrophages are considered to be responsible for initiating the inflammatory cascade in the lung, the airway epithelial NF-κB activation is sufficient to promote neutrophilic airway inflammation.67

Telithromycin (TEL) belongs to the ketolide family, which is a new class of 14-membered ring macrolide agents that are characterized by a keto group at position 3 of the macrolide ring.8TEL is active against bacteria causing community-acquired pneumonia, acute exacerbation of chronic bronchitis, and acute sinusitis.910 TEL exerts immunomodulatory effects in vitro1113 and in experimental models of septic shock,1415 but there have been no studies evaluating the activity of TEL in LPS-induced airway inflammation in mice. We hypothesized that the antiinflammatory activity would be an important component of the therapeutic effect of TEL on respiratory infections. Thus, we designed the present study to test the antiinflammatory activity of TEL in an in vitro model of LPS-induced airway inflammation, and to determine whether this antiinflammatory activity occurs in vivo, in a mouse model of acute, LPS-induced lung injury.

Cells and Culture Conditions

Murine lung epithelial (MLE) cell 12 line was provided by Dr. S. Roman-Roman (Prostrakan Pharmaceuticals; Romainville, France). MLE-12 cells were grown in Roswell Park Memorial Institute 1640 medium supplemented with 5% heat-inactivated fetal calf serum (FCS), 1 mmol/L sodium pyruvate, and 2 mmol/L L-glutamine (Sigma; St Louis, MO). Macrophage cell line RAW 264.7 was provided by Dr. F. Gamarro (Lopez Neyra Institute of Parasitology and Biomedicine, CSIC; Granada, Spain). RAW 264.7 cells were cultured in Dulbecco minimal essential medium (DMEM) [Sigma] supplemented with 10% FCS, 1 mmol/L sodium pyruvate, and 2 mmol/L L-glutamine. Both MLE-12 and RAW 264.7 cells were cultured at 37°C in 5% CO2.

In Vitro Model of LPS-Induced Inflammation

RAW 264.7 cells were grown to confluence in 24-well plates, and stimulated with 1 μg/mL LPS. TEL (Aventis Pharma; Neuville-sur-Saone, France) was diluted in the culture medium and added to cultures at final concentrations of 2.5, 5, and 10 μg/mL, respectively. After incubation for 6 or 24 h, cell-free supernatants were collected and stored at − 20°C until assayed.

MLE-12 cells were grown to confluence in 24-well plates and incubated with 2.5, 5, and 10 μg/mL TEL for 1 h. The culture medium was replaced by supernatant from 24-h cultures of RAW 264.7 cells unstimulated and stimulated with LPS. Supernatants were previously diluted 1:2 in fresh medium. After incubation for 24 h, supernatants from MLE-12 cells were removed and frozen until assayed.

DNA Transfection and Luciferase Assay

The plasmid NF-κB containing the luciferase coding sequence under the control of NF-κB was kindly supplied by Dr. S. Roman. Cells were seeded at a cell density of 104 cells per well in 96-well plates and were transfected on the following day with plasmid NF-κB (Lipofectamine 2000; Invitrogen SA; Izasa Barcelona, Spain). To normalize for transfection efficiency, plasmid RL-TK containing the Renilla luciferase coding sequence (Promega; Madison, WI) was cotransfected as an internal control. At 24 h posttransfection, the medium was replaced by DMEM with 0.2% FCS. Cells were exposed to TEL, and 1 h later LPS was added (1 μg/mL). After 24 h of incubation, cells were lysed and assayed with a commercial kit (Dual-Luciferase Reporter Assay System; Promega).

DNA Binding Activity of NF-kB

The DNA-binding activity of NF-kB in nuclear extracts was measured (TransAM kit; Active Motif; Carlsbad, CA), according to the instructions of the manufacturer.

Apoptosis Assay

The effect of TEL in the rate of apoptosis in RAW 264.7 and MLE-12 cells was assessed (Cell Death Detection ELISA PLUS System; Roche Diagnostics; Mannheim, Germany). Cells were cultured for 24 h at a density of 5 × 103 cells per well in 96-well plates. Culture media were replaced by DMEM supplemented with 1% FCS, and some cultures received 10 μg/mL TEL 1 h before the induction of apoptosis. RAW 264.7 cells were induced with LPS for 24 h, or with camptothecin (2 μg/mL) [Sigma] for 5 h. MLE-12 cells were induced for 24 h with supernatants from unstimulated and LPS-stimulated RAW 264.7 cells, or with camptothecin. Cells were washed with phosphate-buffered saline (PBS) solution and processed according to the instructions of the manufacturer.

Mouse Model of LPS-Induced Lung Injury

Female BALB/c mice, 10 to 12 weeks old, were obtained (Technical Services of the University of Granada; Granada, Spain). The experiments were approved and supervised by the local ethics committee at the University of Granada. Animals were exposed for 20 min to an aerosol of LPS (500 μg/mL) using a custom-built chamber that was directly connected to an air nebulizer (Miko; CA-MI snc; Parma, Italy) that produces particles in the range of 1 to 5 μm at an air flow of 7 L/min. At 4 and 24 h after LPS exposure, the mice were killed, and the trachea was exposed by a midline incision and cannulated with a sterile polyethylene tube attached to a 1-mL syringe. BAL was performed twice in a total volume of 1 mL of PBS solution.

Analysis of Leukocytes in BAL

BAL fluid was centrifuged at 4°C at 1,000 revolutions per minute, and the supernatants were stored at − 20°C for cytokine, nitrite, and proteins assays. Cell pellets were resuspended in PBS solution containing 1% (w/v) bovine serum albumin (Sigma) for total cell counting in a hemocytometer, and macrophage and granulocytes populations were counted by flow cytometry using rat monoclonal antibodies (BD Pharmingen; San Diego, CA). The monoclonal antibodies used were an R-phycoerythrin- conjugated anti-CD14 to stain macrophages and a fluorescein isothiocyanate-conjugated Gr-1 (Ly-6G) to stain neutrophils. Cell samples were analyzed by using a flow cytometer (FACS-Vantage; Becton Dickinson; Mountain View, CA) with software for data acquisition (CELL Quest; Becton Dickinson). Data for 10,000 cells were acquired.

Cytokine Assay

Cytokines were quantified by commercially available enzyme immunoassays (TNF-α; Pierce; Rockford, IL; and macrophage inflammatory protein [MIP]-2; R&D Systems; Minneapolis, MN). The cytokine concentrations were interpolated from the standard curves for recombinant cytokines.

Nitrite Assay

Nitrite concentrations were quantified by a standard Griess reaction adapted to microplates.16 The absorbance at 550 nm was determined with reference to a sodium nitrite standard curve.

Statistical Analysis

The significance of the differences between the means was tested by a Student t test. A p value of < 0.05 was considered to be statistically significant.

Effect of TEL on Cytokine Production by LPS-Stimulated RAW 264.7 Cells and by MLE-12 Cells Stimulated With Supernatants From RAW 264.7 Cultures

The macrophage-epithelial interaction was recreated to provide a model of the pulmonary acute inflammation. Previously, we checked that the cellular metabolic activity17 of the RAW 264.7 and MLE-12 cells was not affected by TEL (10 μg/mL; data not shown). Next, RAW 264.7 macrophages were stimulated with LPS for 24 h to measure the production of MIP-2. Pretreatment with TEL 1 h before LPS stimulation suppressed in a dose-dependent manner the production of the chemokine. The levels of inhibition were as follows for TEL administration when compared with TEL-free cultures (Fig 1 , top, A): 2.5 μg/mL, 17% (p < 0.05); 5 μg/mL, 19% (p < 0.01); and 10 μg/mL, 26% (p < 0.005). Direct stimulation with LPS did not induce the production of MIP-2 by MLE-12 cells (data not shown), but the incubation of MLE-12 cells with unstimulated or LPS-stimulated RAW 264.7 supernatants demonstrated that macrophage-derived inflammatory mediators induced the production of MIP-2 by the epithelial cells (Fig 1, bottom, B). As depicted in Figure 1, bottom, B, MIP-2 production by MLE-12 cells incubated for 24 h with supernatants from LPS-treated RAW 264.7 macrophages was also suppressed by 14% (p < 0.05) at 2.5 μg/mL of TEL, 30% (p < 0.05) at 5 μg/mL of TEL, and 36% (p < 0.005) at 10 μg of TEL.

RAW 264.7 macrophages produced TNF-α in response to LPS stimulation, and this production was significantly inhibited by 24% (p < 0.05), 36% (p < 0.05), and 54% (p < 0.005) 6 h after stimulation with LPS in cultures pretreated with 2.5, 5, and 10 μg/mL of TEL, respectively, 1 h before stimulation (Fig 2 , top, A). Suppression persisted for 24 h after stimulation (data not shown). The inhibition was also observed when TEL and LPS were simultaneously added, with suppression levels of 12% (p < 0.05), 30% (p < 0.01), and 48% (p < 0.005) obtained with 2.5, 5, and 10 μg/mL of TEL, respectively (Fig 2, bottom, B).

Effect of TEL on the Activation of NF-κB in RAW 264.7 Macrophages and MLE-12 Cells

We measured the effect of TEL on NF-κB DNA-binding activity by enzyme-linked immunosorbent assay in nuclear extracts from cells 1 h after stimulation with LPS-containing or TNF-α-containing supernatants. Pretreatment with TEL was performed 1 h before stimulation. Following the exposure of RAW 264.7 macrophages to 2.5, 5, and 10 μg/mL ketolide, NF-κB DNA-binding activity was inhibited by 26% (p < 0.05), 34% (p < 0.01), and 41% (p < 0.005), respectively (Fig 3 , top, A). Similarly, pretreatment with TEL significantly reduced NF-κB DNA-binding activity in MLE-12 epithelial cells that were incubated for 1 h with supernatants from LPS-treated RAW 264.7 macrophages. The inhibition levels were 23% (p < 0.05) at 5 μg/mL TEL and 30% (p < 0.05) at 10 μg/mL TEL (Fig 3, bottom, B).

The inhibitory activity of TEL on NF-κB was confirmed in experiments with cells that were transitorily transfected with a plasmid containing the luciferase coding sequence under the control of this transcription factor. Transfected RAW 264.7 macrophages were stimulated with LPS for 24 h. TEL was added 1 h before stimulation. NF-κB activation was inhibited by 24% (p < 0.05) with administration of 2.5 μg/mL TEL, 37% (p < 0.01) with administration of 5 μg/mL TEL, and 61% (p < 0.005) with administration of 10 μg/mL TEL (Fig 4 , top, A). On the other hand, transfected MLE-12 cells were incubated for 24 h with supernatants from LPS-treated RAW 264.7 macrophages, and some cultures received TEL 1 h before stimulation. NF-κB activation was inhibited by 25% (p < 0.05) with administration of 2.5 μg/mL TEL, 49% (p < 0.05) with administration of 5 μg/mL TEL, and 61% (p < 0.05) with administration of 10 μg/mL TEL (Fig 4, bottom, B).

Effect of TEL on Apoptosis of RAW 264.7 Macrophages and MLE-12 Cells

RAW 264.7 macrophages were induced with LPS for 24 h or with camptothecin for 5 h, and apoptotic fragmentation of DNA was quantified. Pretreatment with TEL (10 μg/mL) 1 h before induction increased the apoptotic activity induced by LPS by 1.23-fold (p < 0.05) and the apoptotic activity induced by camptothecin (p < 0.05) by 1.34-fold (Fig 5 , top, A). Similarly, apoptotic activity was determined in MLE-12 epithelial cells induced with supernatants from LPS-treated RAW 264.7 macrophages for 24 h or with camptothecin for 5 h. In both cases, the apoptotic activity was increased in TEL-pretreated cultures. The increases were 1.62-fold (p < 0.005) in cells induced with supernatants from LPS-treated RAW 264.7 macrophages, and 1.29-fold (p < 0.005) in LPS-induced cells (Fig 5, bottom, B).

Effects of TEL on the Composition of BAL in LPS-Nebulized Mice

We analyzed the effect of a single intraperitoneal (IP) dose of TEL (20 mg/kg) on the recruitment of cells in lungs after LPS aerosol exposure. As shown in Figure 6 , the inhalation of LPS elicited a massive accumulation of leukocytes in BAL fluid, and flow cytometry analysis revealed a major contribution of neutrophils among the accumulated cells. TEL pretreatment (1 h before LPS stimulation) induced a significant reduction in both total cell counts and neutrophil counts at 4 h after LPS exposure (p < 0.05 in both cases) [Fig 6, top, A]; the variations in macrophages were negligible. A similar picture was observed at 24 h after nebulization (p < 0.005 in both cases) [Fig 6, bottom, B].

Untreated controls and mice that received TEL, LPS, or TEL plus LPS did not differ significantly in terms of the protein levels found in BAL fluid during the first 4 h after LPS exposure; however, at 24 h after LPS there was a dramatic increase in protein concentration, which was reduced by 25% (p < 0.01) in TEL-pretreated mice (Fig 7 , top, A). Similarly, when BAL fluid was collected at 24 h after LPS exposure, the concentrations of nitrite (a stable metabolite of the nitric oxide [NO] produced by iNOS) was reduced by 20% (p < 0.05) as a consequence of the pretreatment with TEL (Fig 7, bottom, B).

We also measured the levels of MIP-2 and TNF-α in BAL fluid samples. The levels of MIP-2 markedly rose at 4 h after LPS exposure and dropped at 24 h; in both cases, pretreatment with TEL induced significant reductions (38% [p < 0.05] and 23% [p < 0.05], respectively) [Fig 8 , top, A]. The levels of TNF-α showed a similar kinetic, and were reduced by 49% (p < 0.005) and 60% (p < 0.005) in BAL fluid samples from TEL-pretreated mice that were killed at 4 and 24 h, respectively, after LPS exposure (Fig 8, bottom, B).

Recreating the macrophage-epithelial interaction in vitro provides a useful model of the start of lung inflammation.1819 Our results demonstrate that macrophages respond directly to LPS, whereas epithelial cells respond to LPS-induced monokines. A similar sequence has been described by other authors.18 When activated, both macrophages and epithelial cells produce a number of proinflammatory mediators, including neutrophil-recruiting chemokines such as MIP-2. In the present study, we found that TEL down-regulated the production of MIP-2 by LPS-stimulated RAW 264.7 macrophages and by monokine-stimulated MLE-12 cells. TEL also reduced the release of TNF-α by LPS-stimulated macrophages. This result is relevant since TNF-α is considered to be a pivotal mediator that triggers the production of MIP-2 in epithelial cells.18,20 Thus, TEL seems to exert antiinflammatory effects at the following two levels: (1) by inhibiting TNF-α production by inflammatory macrophages; and (2) by inhibiting NF-κB activation in TNF-α-stimulated epithelial cells.

The transcription factor NF-κB plays a crucial role in the inflammatory response, and the activation of NF-κB was inhibited by TEL in both LPS-stimulated RAW 264.7 macrophages and monokine-stimulated MLE-12 cells. The activation of NF-κB results in the expression of genes encoding inflammation mediators such as TNF-α21and MIP-2.22 Therefore, our data suggest that TEL inhibited the production of both molecules through the inhibition of NF-κB DNA binding.

TEL was not an apoptosis inducer, but enhanced apoptosis in LPS-stimulated RAW 264.7 macrophages and monokine-stimulated MLE-12 cells. Following their activation, macrophages and neutrophils undergo apoptosis as a mechanism to limit the ability of these inflammatory cells to damage tissue. Phagocytosis of apoptotic cells by macrophages actively suppresses the release of proinflammatory mediators and induces an antiinflammatory state.2324 Our findings suggest that TEL could inhibit the production of TNF-α and MIP-2 not only via NF-κB inhibition, but also through the induction of apoptosis. NF-κB inhibition by TEL was observed as soon as 1 h after cell stimulation, and the decrease in TNF-α production by RAW 264.7 macrophages was significant at 6 h after stimulation with LPS. Moreover, it has been described25 that inflammatory cells are removed by apoptosis at the end of the inflammatory response. Therefore, TEL likely acts in the early phase of the inflammatory process by reducing the release of inflammatory mediators through NF-κB inhibition, and in the later phase through the enhancement of inflammatory cell apoptosis.

The stimulation of macrophages with LPS promotes apoptosis through both NO-dependent and NO- independent pathways.2627 Apoptosis in MLE-12 cells may be triggered by TNF-α and by the NO present in supernatants from LPS-stimulated RAW 264.7 macrophages. Other agents with antiinflammatory activity and the capacity to inhibit the activation of NF-κB are also able to promote apoptosis in inflammatory cells.2831

The in vivo intratracheal administration of LPS has been accepted as a clinically relevant model of severe lung inflammation.32 Aerosol exposure of mice to LPS produced an early wave of the MIP-2 chemokine in BAL fluid at 4 h after LPS exposure. This correlated with a massive recruitment of neutrophils into the airways, which was detectable at 4 h and persisted for 24 h following LPS exposure. The pretreatment of mice with TEL significantly reduced MIP-2 levels and neutrophil influx in BAL fluid. In addition, the LPS-induced wave of TNF-α was also inhibited by TEL.

Mice exposed to LPS presented with a high protein content in BAL fluid, attesting to the development of pulmonary edema.32A significant improvement in lung edema was produced by TEL at 24 h post-LPS challenge. This effect might be related to the inhibition of neutrophil recruitment, because neutrophils are considered as the primary cellular effectors of alveolocapillary damage in patients with acute lung injury.33 Moreover, TEL significantly suppressed the local activation of iNOS, as revealed by the decreased levels of nitrite in BAL fluid obtained from TEL-pretreated mice.

The data presented in this study demonstrate that TEL exerts marked antiinflammatory effects on LPS-induced airways inflammation. The following two mechanisms for this are proposed: TEL decreases the production of proinflammatory cytokines and inflammatory mediators from macrophages and epithelial cells by inhibiting NF-κB; and TEL increases apoptosis in activated macrophages and epithelial cells. Both mechanisms have been described in structurally related macrolides. Azithromycin reduces the in vitro production of TNF-α through the inhibition of NF-κB DNA binding.34Clarithromycin35and roxithromycin36reduce the in vivo production of TNF-α and other inflammatory mediators. The apoptosis of activated lymphocytes is augmented by roxithromycin,37 clarithromycin, and azithromycin,38whereas erythromycin accelerates apoptosis in neutrophils.39 In conclusion, TEL is a ketolide antibiotic that shares antiinflammatory properties with macrolides, which appears to be an important component of the therapeutic effect on respiratory infections.

Abbreviations: DMEM = Dulbecco modified essential medium; FCS = fetal calf serum; iNOS = inducible nitric oxide synthase; IP = intraperitoneal; LPS = lipopolysaccharide; MIP = macrophage inflammatory protein; MLE = murine lung epithelial; NF = nuclear factor; NO = nitric oxide; PBS = phosphate-buffered saline; TEL = telithromycin; TNF = tumor necrosis factor

Support for this research was provided by Junta de Andalucía (Research Group CVI201). Dr. Leiva was the recipient of a grant from the Ministerio de Educación, Cultura y Deporte.

The authors have reported to the ACCP that no significant conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Figure Jump LinkFigure 1. Inhibitory effect of TEL on an in vitro model of airway inflammation. Top, A: MIP-2 production by unstimulated (solid bars) and LPS-stimulated (open bars) RAW 264.7 cells. Bottom, B: MIP-2 production by MLE-12 cells incubated with supernatants from unstimulated RAW 264.7 cultures (open bars) or from LPS-stimulated RAW 264.7 cultures (solid bars). MIP-2 concentrations present in RAW 264.7 supernatants were subtracted from MIP-2 levels in MLE-12 cultures. Cells were stimulated with LPS for 24 h, and TEL was added 1 h before stimulation. The results are reported as the means of data from three cultures. Lines represent the SD of the means. TEL-containing cultures were compared with the respective TEL-free culture by Student t test, as follows: * = p < 0.05; ** = p < 0.01; *** = p < 0.005.Grahic Jump Location
Figure Jump LinkFigure 2. Inhibition by TEL of TNF-α production by unstimulated (solid bars) and LPS-stimulated (open bars) RAW 264.7 cells. Cells were treated with LPS for 6 h. Top, A: TEL was added 1 h before LPS. Bottom, B: TEL and LPS were simultaneously added. The results are reported as the means of data from three cultures. Lines represent the SD of the means. TEL-containing cultures were compared with the respective TEL-free culture by Student t test, as follows: * = p < 0.05; ** = p < 0.01; *** = p < 0.005.Grahic Jump Location
Figure Jump LinkFigure 3. The inhibitory effect of TEL on NF-κB DNA-binding activity as measured by enzyme-linked immunosorbent assay in nuclear extracts from RAW 264.7 and MLE-12 cells. DNA binding of NF-κB (p65) was examined in nuclear extracts by a commercial immunoassay as described in the “Material and Methods” section. Top, A: NF-κB activation in unstimulated (solid bars) and LPS-stimulated (open bars) RAW 264.7 cells. Bottom, B: NF-κB activation in MLE-12 cells incubated with supernatants from unstimulated RAW 264.7 cultures (open bars) or from LPS-stimulated RAW 264.7 cultures (solid bars). The results are reported as the means of data from three cultures. Lines represent the SD of the means. TEL-containing cultures were compared with the respective TEL-free culture by Student t test, as follows: * = p < 0.05; ** = p < 0.01; *** = p < 0.005.Grahic Jump Location
Figure Jump LinkFigure 4. The inhibitory effect of TEL on the activation of NF-κB in RAW 264.7 and MLE-12 cells transitorily transfected with a NF-κB luciferase reporter construct. Top, A: NF-κB activation in unstimulated (solid bars) and LPS-stimulated (open bars) RAW 264.7 cells. Bottom, B: NF-κB activation in MLE-12 cells incubated with supernatants from unstimulated RAW 264.7 cultures (open bars) or from LPS-stimulated RAW 264.7 cultures (solid bars). Results are the means of data from three cultures. Lines represent the SD of the means. TEL-containing cultures were compared with the respective TEL-free culture by Student t test, as follows: * = p < 0.05; ** = p < 0.01; *** = p < 0.005.Grahic Jump Location
Figure Jump LinkFigure 5. The effect of TEL on apoptosis in an in vitro model of airway inflammation. Monosomes and oligonucleosomes generated during the apoptotic fragmentation of cellular DNA were quantified by a commercial immunoassay as described in the “Material and Methods” section. Top, A: LPS-induced and camptothecin-induced apoptosis in RAW 264.7 cells incubated without TEL (solid bars) or with TEL (10 μg/mL) [open bars]. Bottom, B: apoptosis induced by camptothecin and supernatants from LPS-stimulated RAW 264.7 cells in MLE-12 cells incubated without TEL (solid bars) or with TEL (10 μg/mL) [open bars]. Cells were induced with LPS or supernatants from RAW 264.7 cultures for 24 h, or with camptothecin for 5 h, and TEL was added 1 h before induction. The results are reported as the means of data from three cultures. Lines represent the SD of the means. TEL-containing cultures were compared with the respective TEL-free culture by Student t test, as follows: * = p < 0.05; ** = p < 0.005.Grahic Jump Location
Figure Jump LinkFigure 6. The effect of TEL on the cellular composition of BAL fluid in an in vivo model of LPS-induced acute lung injury. Top, A: BAL fluid samples were collected at 4 h after the inhalation of LPS. Bottom, B: BAL fluid samples were collected at 24 h after the inhalation of LPS. Untreated controls (open bars) and TEL-treated mice (dotted bars) were exposed to an aerosol of PBS solution alone; nebulizer concentration for mice treated with LPS alone (gray bars) and with TEL and LPS (solid bars) was 500 μg/mL. TEL pretreatment (20 mg/kg) was administered by the IP route 1 h before nebulization. The absolute numbers of macrophages and neutrophils were obtained as the product of flow cytometry percentage and hemocytometer total leukocyte counts. The results are reported as the means of data from eight animals. Lines represent the SD of the means. TEL-pretreated groups were compared with the respective TEL-free groups by Student t test, as follows: * = p < 0.05; ** = p < 0.005.Grahic Jump Location
Figure Jump LinkFigure 7. The effect of TEL on BAL fluid concentrations of proteins (top, A) and nitrite (bottom, B) in an in vivo model of LPS-induced acute lung injury. Untreated controls (open bars) and TEL-treated mice (dotted bars) were exposed to an aerosol of PBS solution alone; the nebulizer concentrations for mice treated with LPS alone (gray bars) and with TEL and LPS (solid bars) were 500 μg/mL. TEL pretreatment (20 mg/kg) was administered by the IP route 1 h before nebulization. The results are reported as the means of data from four animals. Lines represent the SD of the means. TEL-pretreated groups were compared with the respective TEL-free groups by Student t test, as follows: * = p < 0.05; ** = p < 0.01.Grahic Jump Location
Figure Jump LinkFigure 8. The effect of TEL on BAL fluid concentrations of MIP-2 (top, A) and TNF-α (bottom, B) in an in vivo model of LPS-induced acute lung injury. Untreated controls (open bars) and TEL-treated mice (dotted bars) were exposed to an aerosol of PBS solution alone; the nebulizer concentrations for mice treated with LPS alone (gray bars) and with TEL and LPS (solid bars) were 500 μg/mL. TEL pretreatment (20 mg/kg) was administered by the IP route 1 h before nebulization. The results are reported as the means of data from four animals. Lines represent the SD of the means. TEL-pretreated groups were compared with the respective TEL-free groups by Student t test, as follows: * = p < 0.05; ** = p < 0.005.Grahic Jump Location
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Fadok, VA, Bratton, DL, Henson, PM Phagocyte receptors for apoptotic cells: recognition, uptake, and consequences.J Clin Invest2001;108,957-962
 
Tassiulas, I, Park-Min, KH, Hu, Y, et al Apoptotic cells inhibit LPS-induced cytokine and chemokine production and IFN responses in macrophages.Hum Immunol2007;68,156-164
 
Savill, J Apoptosis in resolution of inflammation.J Leukoc Biol1997;61,375-380
 
Terenzi, F, Diaz-Guerra, MJ, Casado, M, et al Bacterial lipopeptides induce nitric oxide-independent pathways in rat macrophages.J Biol Chem1995;270,6017-6021
 
Bosca, L, Hortelano, S Mechanisms of nitric oxide-dependent apoptosis: involvement of mitochondrial mediators.Cell Signal1999;11,239-244
 
Ward, C, Chilvers, ER, Lawson, MF, et al NF-κB activation is a critical regulator of human granulocyte apoptosisin vitro.J Biol Chem1999;274,4309-4318
 
Hortelano, S, Castrillo, A, Alvarez, AM, et al Contribution of cyclopentenone prostraglandins to the resolution of inflammation through the potentiation of apoptosis in activated macrophages.J Immunol2000;165,6525-6531
 
Fitzpatrick, LR, Wang, J, Le, T Caffeic acid phenethyl ester, an inhibitor of nuclear factor-kB, attenuates bacterial peptidoglycan polysaccharide-induced colitis in rats.J Pharmacol Exp Ther2001;299,915-920
 
Negrotto, S, Malaver, E, Alvarez, ME, et al Aspirin and salicylate suppress polymorphonuclear apoptosis delay mediated by proinflammatory stimuli.J Pharmacol Exp Ther2006;319,972-979
 
Llaudet, L, Mabley, JG, Pacher, P, et al Inosine exerts a broad range of antiinflammatory effects in a murine model of acute lung injury.Ann Surg2002;235,568-578
 
Heflin, AC, Brigham, KL Prevention by granulocyte depletion of increased vascular permeability of sheep lung following endotoxemia.J Clin Invest1981;68,1253-1260
 
Cigana, C, Assael, BM, Melotti, P Azithromycin selectively erduces tumor necrosis factor alpha levels in cystic fibrosis airway epithelial cells.Antimicrob Agents Chemother2007;51,975-981
 
Hardy, RD, Rios, AM, Chavez-Bueno, S, et al Antimicrobial and immunologic activities of clarithromycin in a murine model ofMycoplasma pneumoniae-induced pneumonia.Antimicrob Agents Chemother2003;47,1614-1620
 
Urasaki, Y, Nori, M, Iwata, S, et al Roxithromycin specifically inhibits development of collagen induced arthritis and production of proinflammatory cytokines by human T cells and macrophages.J Rheumatol2005;32,1765-1774
 
Ogawa, N, Sugawara, Y, Fujiwara, Y, et al Roxithromycin promotes lymphocyte apoptosis in Dermatophagoides-sensitive asthma patients.Eur J Pharmacol2003;474,273-281
 
Kadota, JI, Mizunoe, S, Kishi, K, et al Antibiotic-induced apoptosis in human activated peripheral lymphocytes.Int J Antimicrob Agents2005;25,216-220
 
Aoshiba, K, Nagal, A, Konno, K Erythromycin shortens neutrophil survival by accelerating apoptosis.Antimicrob Agents Chemother1995;39,872-877
 

Figures

Figure Jump LinkFigure 1. Inhibitory effect of TEL on an in vitro model of airway inflammation. Top, A: MIP-2 production by unstimulated (solid bars) and LPS-stimulated (open bars) RAW 264.7 cells. Bottom, B: MIP-2 production by MLE-12 cells incubated with supernatants from unstimulated RAW 264.7 cultures (open bars) or from LPS-stimulated RAW 264.7 cultures (solid bars). MIP-2 concentrations present in RAW 264.7 supernatants were subtracted from MIP-2 levels in MLE-12 cultures. Cells were stimulated with LPS for 24 h, and TEL was added 1 h before stimulation. The results are reported as the means of data from three cultures. Lines represent the SD of the means. TEL-containing cultures were compared with the respective TEL-free culture by Student t test, as follows: * = p < 0.05; ** = p < 0.01; *** = p < 0.005.Grahic Jump Location
Figure Jump LinkFigure 2. Inhibition by TEL of TNF-α production by unstimulated (solid bars) and LPS-stimulated (open bars) RAW 264.7 cells. Cells were treated with LPS for 6 h. Top, A: TEL was added 1 h before LPS. Bottom, B: TEL and LPS were simultaneously added. The results are reported as the means of data from three cultures. Lines represent the SD of the means. TEL-containing cultures were compared with the respective TEL-free culture by Student t test, as follows: * = p < 0.05; ** = p < 0.01; *** = p < 0.005.Grahic Jump Location
Figure Jump LinkFigure 3. The inhibitory effect of TEL on NF-κB DNA-binding activity as measured by enzyme-linked immunosorbent assay in nuclear extracts from RAW 264.7 and MLE-12 cells. DNA binding of NF-κB (p65) was examined in nuclear extracts by a commercial immunoassay as described in the “Material and Methods” section. Top, A: NF-κB activation in unstimulated (solid bars) and LPS-stimulated (open bars) RAW 264.7 cells. Bottom, B: NF-κB activation in MLE-12 cells incubated with supernatants from unstimulated RAW 264.7 cultures (open bars) or from LPS-stimulated RAW 264.7 cultures (solid bars). The results are reported as the means of data from three cultures. Lines represent the SD of the means. TEL-containing cultures were compared with the respective TEL-free culture by Student t test, as follows: * = p < 0.05; ** = p < 0.01; *** = p < 0.005.Grahic Jump Location
Figure Jump LinkFigure 4. The inhibitory effect of TEL on the activation of NF-κB in RAW 264.7 and MLE-12 cells transitorily transfected with a NF-κB luciferase reporter construct. Top, A: NF-κB activation in unstimulated (solid bars) and LPS-stimulated (open bars) RAW 264.7 cells. Bottom, B: NF-κB activation in MLE-12 cells incubated with supernatants from unstimulated RAW 264.7 cultures (open bars) or from LPS-stimulated RAW 264.7 cultures (solid bars). Results are the means of data from three cultures. Lines represent the SD of the means. TEL-containing cultures were compared with the respective TEL-free culture by Student t test, as follows: * = p < 0.05; ** = p < 0.01; *** = p < 0.005.Grahic Jump Location
Figure Jump LinkFigure 5. The effect of TEL on apoptosis in an in vitro model of airway inflammation. Monosomes and oligonucleosomes generated during the apoptotic fragmentation of cellular DNA were quantified by a commercial immunoassay as described in the “Material and Methods” section. Top, A: LPS-induced and camptothecin-induced apoptosis in RAW 264.7 cells incubated without TEL (solid bars) or with TEL (10 μg/mL) [open bars]. Bottom, B: apoptosis induced by camptothecin and supernatants from LPS-stimulated RAW 264.7 cells in MLE-12 cells incubated without TEL (solid bars) or with TEL (10 μg/mL) [open bars]. Cells were induced with LPS or supernatants from RAW 264.7 cultures for 24 h, or with camptothecin for 5 h, and TEL was added 1 h before induction. The results are reported as the means of data from three cultures. Lines represent the SD of the means. TEL-containing cultures were compared with the respective TEL-free culture by Student t test, as follows: * = p < 0.05; ** = p < 0.005.Grahic Jump Location
Figure Jump LinkFigure 6. The effect of TEL on the cellular composition of BAL fluid in an in vivo model of LPS-induced acute lung injury. Top, A: BAL fluid samples were collected at 4 h after the inhalation of LPS. Bottom, B: BAL fluid samples were collected at 24 h after the inhalation of LPS. Untreated controls (open bars) and TEL-treated mice (dotted bars) were exposed to an aerosol of PBS solution alone; nebulizer concentration for mice treated with LPS alone (gray bars) and with TEL and LPS (solid bars) was 500 μg/mL. TEL pretreatment (20 mg/kg) was administered by the IP route 1 h before nebulization. The absolute numbers of macrophages and neutrophils were obtained as the product of flow cytometry percentage and hemocytometer total leukocyte counts. The results are reported as the means of data from eight animals. Lines represent the SD of the means. TEL-pretreated groups were compared with the respective TEL-free groups by Student t test, as follows: * = p < 0.05; ** = p < 0.005.Grahic Jump Location
Figure Jump LinkFigure 7. The effect of TEL on BAL fluid concentrations of proteins (top, A) and nitrite (bottom, B) in an in vivo model of LPS-induced acute lung injury. Untreated controls (open bars) and TEL-treated mice (dotted bars) were exposed to an aerosol of PBS solution alone; the nebulizer concentrations for mice treated with LPS alone (gray bars) and with TEL and LPS (solid bars) were 500 μg/mL. TEL pretreatment (20 mg/kg) was administered by the IP route 1 h before nebulization. The results are reported as the means of data from four animals. Lines represent the SD of the means. TEL-pretreated groups were compared with the respective TEL-free groups by Student t test, as follows: * = p < 0.05; ** = p < 0.01.Grahic Jump Location
Figure Jump LinkFigure 8. The effect of TEL on BAL fluid concentrations of MIP-2 (top, A) and TNF-α (bottom, B) in an in vivo model of LPS-induced acute lung injury. Untreated controls (open bars) and TEL-treated mice (dotted bars) were exposed to an aerosol of PBS solution alone; the nebulizer concentrations for mice treated with LPS alone (gray bars) and with TEL and LPS (solid bars) were 500 μg/mL. TEL pretreatment (20 mg/kg) was administered by the IP route 1 h before nebulization. The results are reported as the means of data from four animals. Lines represent the SD of the means. TEL-pretreated groups were compared with the respective TEL-free groups by Student t test, as follows: * = p < 0.05; ** = p < 0.005.Grahic Jump Location

Tables

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Fadok, VA, Bratton, DL, Henson, PM Phagocyte receptors for apoptotic cells: recognition, uptake, and consequences.J Clin Invest2001;108,957-962
 
Tassiulas, I, Park-Min, KH, Hu, Y, et al Apoptotic cells inhibit LPS-induced cytokine and chemokine production and IFN responses in macrophages.Hum Immunol2007;68,156-164
 
Savill, J Apoptosis in resolution of inflammation.J Leukoc Biol1997;61,375-380
 
Terenzi, F, Diaz-Guerra, MJ, Casado, M, et al Bacterial lipopeptides induce nitric oxide-independent pathways in rat macrophages.J Biol Chem1995;270,6017-6021
 
Bosca, L, Hortelano, S Mechanisms of nitric oxide-dependent apoptosis: involvement of mitochondrial mediators.Cell Signal1999;11,239-244
 
Ward, C, Chilvers, ER, Lawson, MF, et al NF-κB activation is a critical regulator of human granulocyte apoptosisin vitro.J Biol Chem1999;274,4309-4318
 
Hortelano, S, Castrillo, A, Alvarez, AM, et al Contribution of cyclopentenone prostraglandins to the resolution of inflammation through the potentiation of apoptosis in activated macrophages.J Immunol2000;165,6525-6531
 
Fitzpatrick, LR, Wang, J, Le, T Caffeic acid phenethyl ester, an inhibitor of nuclear factor-kB, attenuates bacterial peptidoglycan polysaccharide-induced colitis in rats.J Pharmacol Exp Ther2001;299,915-920
 
Negrotto, S, Malaver, E, Alvarez, ME, et al Aspirin and salicylate suppress polymorphonuclear apoptosis delay mediated by proinflammatory stimuli.J Pharmacol Exp Ther2006;319,972-979
 
Llaudet, L, Mabley, JG, Pacher, P, et al Inosine exerts a broad range of antiinflammatory effects in a murine model of acute lung injury.Ann Surg2002;235,568-578
 
Heflin, AC, Brigham, KL Prevention by granulocyte depletion of increased vascular permeability of sheep lung following endotoxemia.J Clin Invest1981;68,1253-1260
 
Cigana, C, Assael, BM, Melotti, P Azithromycin selectively erduces tumor necrosis factor alpha levels in cystic fibrosis airway epithelial cells.Antimicrob Agents Chemother2007;51,975-981
 
Hardy, RD, Rios, AM, Chavez-Bueno, S, et al Antimicrobial and immunologic activities of clarithromycin in a murine model ofMycoplasma pneumoniae-induced pneumonia.Antimicrob Agents Chemother2003;47,1614-1620
 
Urasaki, Y, Nori, M, Iwata, S, et al Roxithromycin specifically inhibits development of collagen induced arthritis and production of proinflammatory cytokines by human T cells and macrophages.J Rheumatol2005;32,1765-1774
 
Ogawa, N, Sugawara, Y, Fujiwara, Y, et al Roxithromycin promotes lymphocyte apoptosis in Dermatophagoides-sensitive asthma patients.Eur J Pharmacol2003;474,273-281
 
Kadota, JI, Mizunoe, S, Kishi, K, et al Antibiotic-induced apoptosis in human activated peripheral lymphocytes.Int J Antimicrob Agents2005;25,216-220
 
Aoshiba, K, Nagal, A, Konno, K Erythromycin shortens neutrophil survival by accelerating apoptosis.Antimicrob Agents Chemother1995;39,872-877
 
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