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Baclofen, a GABABR agonist, ameliorates immune-complex mediated acute lung injury by modulating pro-inflammatory mediators.

Jin S, Merchant ML, Ritzenthaler JD, McLeish KR, Lederer ED, Torres-Gonzalez E, Fraig M, Barati MT, Lentsch AB, Roman J, Klein JB, Rane MJ - PLoS ONE (2015)

Bottom Line: ALI increased total protein, tumor necrosis factor α (TNF-α and interleukin-1 receptor associated protein (IL-1R AcP), in the bronchoalveolar lavage fluid (BALF).Protective effects of baclofen treatment on ALI were possibly mediated by inhibition of TNF-α- and IL-1β-mediated inflammatory signaling.GABABR2 agonists may play a potential therapeutic role in ALI.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Louisville, Louisville, Kentucky, United States of America.

ABSTRACT
Immune-complexes play an important role in the inflammatory diseases of the lung. Neutrophil activation mediates immune-complex (IC) deposition-induced acute lung injury (ALI). Components of gamma amino butyric acid (GABA) signaling, including GABA B receptor 2 (GABABR2), GAD65/67 and the GABA transporter, are present in the lungs and in the neutrophils. However, the role of pulmonary GABABR activation in the context of neutrophil-mediated ALI has not been determined. Thus, the objective of the current study was to determine whether administration of a GABABR agonist, baclofen would ameliorate or exacerbate ALI. We hypothesized that baclofen would regulate IC-induced ALI by preserving pulmonary GABABR expression. Rats were subjected to sham injury or IC-induced ALI and two hours later rats were treated intratracheally with saline or 1 mg/kg baclofen for 2 additional hours and sacrificed. ALI was assessed by vascular leakage, histology, TUNEL, and lung caspase-3 cleavage. ALI increased total protein, tumor necrosis factor α (TNF-α and interleukin-1 receptor associated protein (IL-1R AcP), in the bronchoalveolar lavage fluid (BALF). Moreover, ALI decreased lung GABABR2 expression, increased phospho-p38 MAPK, promoted IκB degradation and increased neutrophil influx in the lung. Administration of baclofen, after initiation of ALI, restored GABABR expression, which was inhibited in the presence of a GABABR antagonist, CGP52432. Baclofen administration activated pulmonary phospho-ERK and inhibited p38 MAPK phosphorylation and IκB degradation. Additionally, baclofen significantly inhibited pro-inflammatory TNF-α and IL-1βAcP release and promoted BAL neutrophil apoptosis. Protective effects of baclofen treatment on ALI were possibly mediated by inhibition of TNF-α- and IL-1β-mediated inflammatory signaling. Interestingly, GABABR2 expression was regulated in the type II pneumocytes in lung tissue sections from lung injured patients, further suggesting a physiological role for GABABR2 in the repair process of lung damage. GABABR2 agonists may play a potential therapeutic role in ALI.

No MeSH data available.


Related in: MedlinePlus

Baclofen ameliorates Immunoglobulin G-immune complex (IC) deposition induced acute lung injury (ALI).(A) Fluorescence of FITC-BSA was measured in BALF samples from three animal groups: Control, lung injury (LI), or LI followed by baclofen administration (LI+B). Data is expressed as mean relative fluorescent units ± SEM, n = 7 animals per group. (B) Total protein was measured in BALF samples from three animal groups: control, lung injury (LI), LI followed by baclofen (LI+B). Data are expressed as mean ± SEM, n = 7 animals per group. (C) Bronchoalveolar lavage cells collected from two animal groups: lung injury (LI) (lane 1), or LI followed by baclofen administration (LI+B) (lanes 2 and 3), were lysed and proteins were separated by SDS-PAGE followed by immunoblot analysis for cleaved caspase-3. A representative immunoblot from four independent experiments performed with 4 animals per group. (D) We have quantitated caspase-3 cleavage data from 4 independent experiments using 4 animals per group. LI promoted neutrophil survival while baclofen treatment after initiating LI induced BAL neutrophil apoptosis. (E) This is a representative image of Hematoxylin-Eosin (H&E) staining that was performed on lung tissue sections from 3 animal groups: control, LI and LI +B with 4 animals in control group and 8 animals each in LI and LI+B groups. Magnification 20X. IC deposition significantly increased lung injury score while baclofen treatment after initiating ALI significantly inhibited the lung injury score (bottom right panel). (E) This is a representative image of Naphthol AS-D chloroacetate esterase staining that was performed on lung sections from 3 animal groups: control, LI and LI+B with 3 animals per group. Magnification 40X.
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pone.0121637.g001: Baclofen ameliorates Immunoglobulin G-immune complex (IC) deposition induced acute lung injury (ALI).(A) Fluorescence of FITC-BSA was measured in BALF samples from three animal groups: Control, lung injury (LI), or LI followed by baclofen administration (LI+B). Data is expressed as mean relative fluorescent units ± SEM, n = 7 animals per group. (B) Total protein was measured in BALF samples from three animal groups: control, lung injury (LI), LI followed by baclofen (LI+B). Data are expressed as mean ± SEM, n = 7 animals per group. (C) Bronchoalveolar lavage cells collected from two animal groups: lung injury (LI) (lane 1), or LI followed by baclofen administration (LI+B) (lanes 2 and 3), were lysed and proteins were separated by SDS-PAGE followed by immunoblot analysis for cleaved caspase-3. A representative immunoblot from four independent experiments performed with 4 animals per group. (D) We have quantitated caspase-3 cleavage data from 4 independent experiments using 4 animals per group. LI promoted neutrophil survival while baclofen treatment after initiating LI induced BAL neutrophil apoptosis. (E) This is a representative image of Hematoxylin-Eosin (H&E) staining that was performed on lung tissue sections from 3 animal groups: control, LI and LI +B with 4 animals in control group and 8 animals each in LI and LI+B groups. Magnification 20X. IC deposition significantly increased lung injury score while baclofen treatment after initiating ALI significantly inhibited the lung injury score (bottom right panel). (E) This is a representative image of Naphthol AS-D chloroacetate esterase staining that was performed on lung sections from 3 animal groups: control, LI and LI+B with 3 animals per group. Magnification 40X.

Mentions: To determine the effects of baclofen administration on ALI, three groups of rats were used. A control group received intratracheal saline while BSA with trace amounts of FITC-labeled BSA was administered intravenously. The other two groups received intratracheal anti-BSA while BSA and trace amounts of FITC-labeled BSA were administered intravenousy. Two hr after initiating ALI, one group received intratracheal saline while second group received intratracheal 1 mg/kg baclofen. After additional 2 h rats were killed and bronchoalveolar lavage (BAL) was performed, or lung tissue obtained for histology. Fig 1A shows the BALF fluorescence resulting from leakage of FITC-labeled BSA from blood into the lungs. ALI was associated with a significant increase in BALF fluorescence compared to control that was significantly ameliorated in animals treated with baclofen. The data was averaged and quantitated using 7 animals per group. Fig 1B shows total protein in BALF from control rats and with rats subjected to ALI with or without baclofen administration 2 h after initiating ALI. ALI was associated with significant increase in BALF protein compared to control that was significantly decreased after baclofen treatment. The average number of neutrophils was very low in the control animals (0.13 x 106 ± 0.015 x 106, n = 7 animals). ALI was associated with a significant increase in total neutrophils (6.8 x 106 ± 0.8 x 106, n = 7 animals) that was not significantly altered by administration of baclofen (8.25 x 106 ± 2.05 x 106, n = 7 animals). Thus, baclofen administration significantly reduced the increased vascular permeability and total protein concentrations without affecting neutrophil infiltration in BALF. Next we determined the survival status of cells (neutrophils) in BALF from animals subjected to ALI with or without baclofen treatment using 4 animals per group. We did not include a control lane as no neutrophils are present in the BALF of control animals. BAL cells from animal groups subjected to ALI with or without baclofen were lysed and equal protein amounts were subjected to anti-cleaved caspase-3 (marker of apoptosis) immunoblot analysis. As expected in Fig 1C we see little to no caspase-3 cleavage in BAL neutrophils from lung injured animals (Fig 1C) suggestive of BAL neutrophil survival (delayed neutrophil apoptosis) and more inflammation. In contrast, baclofen treatment after initiating ALI, promoted induction of caspase-3 cleavage (Fig 1C) in BAL neutrophils, suggestive of BAL neutrophil apoptosis and resolution of inflammation. We have quantitated caspase-3 cleavage data from 4 independent experiments performed with 4 animals in each of the two groups tested (Fig 1D). To determine effect of baclofen treatment on IC-induced lung damage, H&E staining was performed on three groups of animals control, lung injured (LI), lung injured followed by baclofen administration (LI+B). Fig 1E shows a representative image of H&E staining on lung tissue sections from three groups of animals tested with 4 animals in control group and 8 animals each in the LI and LI+B groups. H&E staining revealed interstitial and intra-alveolar edema, and hemorrhage after ALI which was markedly inhibited after baclofen administration (Fig 1E). IC deposition significantly increased lung injury score while baclofen treatment after initiating ALI significantly inhibited the lung injury score (Fig 1E).


Baclofen, a GABABR agonist, ameliorates immune-complex mediated acute lung injury by modulating pro-inflammatory mediators.

Jin S, Merchant ML, Ritzenthaler JD, McLeish KR, Lederer ED, Torres-Gonzalez E, Fraig M, Barati MT, Lentsch AB, Roman J, Klein JB, Rane MJ - PLoS ONE (2015)

Baclofen ameliorates Immunoglobulin G-immune complex (IC) deposition induced acute lung injury (ALI).(A) Fluorescence of FITC-BSA was measured in BALF samples from three animal groups: Control, lung injury (LI), or LI followed by baclofen administration (LI+B). Data is expressed as mean relative fluorescent units ± SEM, n = 7 animals per group. (B) Total protein was measured in BALF samples from three animal groups: control, lung injury (LI), LI followed by baclofen (LI+B). Data are expressed as mean ± SEM, n = 7 animals per group. (C) Bronchoalveolar lavage cells collected from two animal groups: lung injury (LI) (lane 1), or LI followed by baclofen administration (LI+B) (lanes 2 and 3), were lysed and proteins were separated by SDS-PAGE followed by immunoblot analysis for cleaved caspase-3. A representative immunoblot from four independent experiments performed with 4 animals per group. (D) We have quantitated caspase-3 cleavage data from 4 independent experiments using 4 animals per group. LI promoted neutrophil survival while baclofen treatment after initiating LI induced BAL neutrophil apoptosis. (E) This is a representative image of Hematoxylin-Eosin (H&E) staining that was performed on lung tissue sections from 3 animal groups: control, LI and LI +B with 4 animals in control group and 8 animals each in LI and LI+B groups. Magnification 20X. IC deposition significantly increased lung injury score while baclofen treatment after initiating ALI significantly inhibited the lung injury score (bottom right panel). (E) This is a representative image of Naphthol AS-D chloroacetate esterase staining that was performed on lung sections from 3 animal groups: control, LI and LI+B with 3 animals per group. Magnification 40X.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4388838&req=5

pone.0121637.g001: Baclofen ameliorates Immunoglobulin G-immune complex (IC) deposition induced acute lung injury (ALI).(A) Fluorescence of FITC-BSA was measured in BALF samples from three animal groups: Control, lung injury (LI), or LI followed by baclofen administration (LI+B). Data is expressed as mean relative fluorescent units ± SEM, n = 7 animals per group. (B) Total protein was measured in BALF samples from three animal groups: control, lung injury (LI), LI followed by baclofen (LI+B). Data are expressed as mean ± SEM, n = 7 animals per group. (C) Bronchoalveolar lavage cells collected from two animal groups: lung injury (LI) (lane 1), or LI followed by baclofen administration (LI+B) (lanes 2 and 3), were lysed and proteins were separated by SDS-PAGE followed by immunoblot analysis for cleaved caspase-3. A representative immunoblot from four independent experiments performed with 4 animals per group. (D) We have quantitated caspase-3 cleavage data from 4 independent experiments using 4 animals per group. LI promoted neutrophil survival while baclofen treatment after initiating LI induced BAL neutrophil apoptosis. (E) This is a representative image of Hematoxylin-Eosin (H&E) staining that was performed on lung tissue sections from 3 animal groups: control, LI and LI +B with 4 animals in control group and 8 animals each in LI and LI+B groups. Magnification 20X. IC deposition significantly increased lung injury score while baclofen treatment after initiating ALI significantly inhibited the lung injury score (bottom right panel). (E) This is a representative image of Naphthol AS-D chloroacetate esterase staining that was performed on lung sections from 3 animal groups: control, LI and LI+B with 3 animals per group. Magnification 40X.
Mentions: To determine the effects of baclofen administration on ALI, three groups of rats were used. A control group received intratracheal saline while BSA with trace amounts of FITC-labeled BSA was administered intravenously. The other two groups received intratracheal anti-BSA while BSA and trace amounts of FITC-labeled BSA were administered intravenousy. Two hr after initiating ALI, one group received intratracheal saline while second group received intratracheal 1 mg/kg baclofen. After additional 2 h rats were killed and bronchoalveolar lavage (BAL) was performed, or lung tissue obtained for histology. Fig 1A shows the BALF fluorescence resulting from leakage of FITC-labeled BSA from blood into the lungs. ALI was associated with a significant increase in BALF fluorescence compared to control that was significantly ameliorated in animals treated with baclofen. The data was averaged and quantitated using 7 animals per group. Fig 1B shows total protein in BALF from control rats and with rats subjected to ALI with or without baclofen administration 2 h after initiating ALI. ALI was associated with significant increase in BALF protein compared to control that was significantly decreased after baclofen treatment. The average number of neutrophils was very low in the control animals (0.13 x 106 ± 0.015 x 106, n = 7 animals). ALI was associated with a significant increase in total neutrophils (6.8 x 106 ± 0.8 x 106, n = 7 animals) that was not significantly altered by administration of baclofen (8.25 x 106 ± 2.05 x 106, n = 7 animals). Thus, baclofen administration significantly reduced the increased vascular permeability and total protein concentrations without affecting neutrophil infiltration in BALF. Next we determined the survival status of cells (neutrophils) in BALF from animals subjected to ALI with or without baclofen treatment using 4 animals per group. We did not include a control lane as no neutrophils are present in the BALF of control animals. BAL cells from animal groups subjected to ALI with or without baclofen were lysed and equal protein amounts were subjected to anti-cleaved caspase-3 (marker of apoptosis) immunoblot analysis. As expected in Fig 1C we see little to no caspase-3 cleavage in BAL neutrophils from lung injured animals (Fig 1C) suggestive of BAL neutrophil survival (delayed neutrophil apoptosis) and more inflammation. In contrast, baclofen treatment after initiating ALI, promoted induction of caspase-3 cleavage (Fig 1C) in BAL neutrophils, suggestive of BAL neutrophil apoptosis and resolution of inflammation. We have quantitated caspase-3 cleavage data from 4 independent experiments performed with 4 animals in each of the two groups tested (Fig 1D). To determine effect of baclofen treatment on IC-induced lung damage, H&E staining was performed on three groups of animals control, lung injured (LI), lung injured followed by baclofen administration (LI+B). Fig 1E shows a representative image of H&E staining on lung tissue sections from three groups of animals tested with 4 animals in control group and 8 animals each in the LI and LI+B groups. H&E staining revealed interstitial and intra-alveolar edema, and hemorrhage after ALI which was markedly inhibited after baclofen administration (Fig 1E). IC deposition significantly increased lung injury score while baclofen treatment after initiating ALI significantly inhibited the lung injury score (Fig 1E).

Bottom Line: ALI increased total protein, tumor necrosis factor α (TNF-α and interleukin-1 receptor associated protein (IL-1R AcP), in the bronchoalveolar lavage fluid (BALF).Protective effects of baclofen treatment on ALI were possibly mediated by inhibition of TNF-α- and IL-1β-mediated inflammatory signaling.GABABR2 agonists may play a potential therapeutic role in ALI.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Louisville, Louisville, Kentucky, United States of America.

ABSTRACT
Immune-complexes play an important role in the inflammatory diseases of the lung. Neutrophil activation mediates immune-complex (IC) deposition-induced acute lung injury (ALI). Components of gamma amino butyric acid (GABA) signaling, including GABA B receptor 2 (GABABR2), GAD65/67 and the GABA transporter, are present in the lungs and in the neutrophils. However, the role of pulmonary GABABR activation in the context of neutrophil-mediated ALI has not been determined. Thus, the objective of the current study was to determine whether administration of a GABABR agonist, baclofen would ameliorate or exacerbate ALI. We hypothesized that baclofen would regulate IC-induced ALI by preserving pulmonary GABABR expression. Rats were subjected to sham injury or IC-induced ALI and two hours later rats were treated intratracheally with saline or 1 mg/kg baclofen for 2 additional hours and sacrificed. ALI was assessed by vascular leakage, histology, TUNEL, and lung caspase-3 cleavage. ALI increased total protein, tumor necrosis factor α (TNF-α and interleukin-1 receptor associated protein (IL-1R AcP), in the bronchoalveolar lavage fluid (BALF). Moreover, ALI decreased lung GABABR2 expression, increased phospho-p38 MAPK, promoted IκB degradation and increased neutrophil influx in the lung. Administration of baclofen, after initiation of ALI, restored GABABR expression, which was inhibited in the presence of a GABABR antagonist, CGP52432. Baclofen administration activated pulmonary phospho-ERK and inhibited p38 MAPK phosphorylation and IκB degradation. Additionally, baclofen significantly inhibited pro-inflammatory TNF-α and IL-1βAcP release and promoted BAL neutrophil apoptosis. Protective effects of baclofen treatment on ALI were possibly mediated by inhibition of TNF-α- and IL-1β-mediated inflammatory signaling. Interestingly, GABABR2 expression was regulated in the type II pneumocytes in lung tissue sections from lung injured patients, further suggesting a physiological role for GABABR2 in the repair process of lung damage. GABABR2 agonists may play a potential therapeutic role in ALI.

No MeSH data available.


Related in: MedlinePlus