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Brief Glutamine Pretreatment Increases Alveolar Macrophage CD163/Heme Oxygenase-1/p38-MAPK Dephosphorylation Pathway and Decreases Capillary Damage but Not Neutrophil Recruitment in IL-1/LPS-Insufflated Rats.

Fernandez-Bustamante A, Agazio A, Wilson P, Elkins N, Domaleski L, He Q, Baer KA, Moss AF, Wischmeyer PE, Repine JE - PLoS ONE (2015)

Bottom Line: Glutamine (GLN) attenuates acute lung injury (ALI) but its effect on alveolar macrophages is unknown.GLN pretreatment before IL-1/LPS also significantly increased HO-1 concentrations and dephosphorylated p38-MAPK levels but not cytokine levels in alveolar macrophages.Short-term GLN pretreatment activates the anti-inflammatory CD163/HO-1/p38-MAPK dephosphorylation pathway of alveolar macrophages and decreases capillary damage but not neutrophil recruitment in IL-1/LPS-insufflated rats.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, University of Colorado SOM, Aurora, Colorado, United States of America; Webb-Waring Center, University of Colorado SOM, Aurora, Colorado, United States of America.

ABSTRACT

Background: Glutamine (GLN) attenuates acute lung injury (ALI) but its effect on alveolar macrophages is unknown. We hypothesized that GLN pretreatment would induce the anti-inflammatory CD163/heme oxygenase (HO)-1/p38-MAPK dephosphorylation pathway in alveolar macrophages and reduce ALI in rats insufflated with interleukin-1 (IL-1) and lipopolysaccharide (LPS).

Methods: Male Sprague-Dawley rats were randomized to the following groups: GLN-IL-1/LPS-, GLN+IL-1/LPS-, GLN-IL-1/LPS+, and GLN+IL-1/LPS+. GLN pretreatment was given via gavage (1 g/kg L-alanyl-L-glutamine) daily for 2 days. ALI was subsequently induced by insufflating 50 ng IL-1 followed by 5mg/kg E.coli LPS. After 24h, bronchoalveolar lavage (BAL) protein, lactate dehydrogenase (LDH) and neutrophil concentrations were analyzed. BAL alveolar macrophage CD163+ expression, HO-1 and p38-MAPK concentrations were measured, as well as alveolar macrophage tumor necrosis factor (TNF)-α and interleukin (IL)-10 concentrations. Histology and immunofluorescence studies were also performed.

Results: Following IL-1/LPS insufflation, GLN pretreated rats had significantly decreased BAL protein and LDH concentrations, but not BAL neutrophil counts, compared to non-GLN pretreated rats. The number of alveolar macrophages and the number of CD163+ macrophages were significantly increased in GLN pretreated IL-1/LPS-insufflated rats compared to non-GLN pretreated, IL-1/LPS-insufflated rats. GLN pretreatment before IL-1/LPS also significantly increased HO-1 concentrations and dephosphorylated p38-MAPK levels but not cytokine levels in alveolar macrophages. Immunofluorescence localized CD163 and HO-1 in alveolar macrophages.

Conclusion: Short-term GLN pretreatment activates the anti-inflammatory CD163/HO-1/p38-MAPK dephosphorylation pathway of alveolar macrophages and decreases capillary damage but not neutrophil recruitment in IL-1/LPS-insufflated rats.

No MeSH data available.


Related in: MedlinePlus

Quantification of CD163+ BAL cells by flow-cytometry.Fig (3a) shows representative flow-cytometry histograms from studied groups. Fluorescence was compared to unstained controls for 35,000 events in each histogram display, and the stained APC-CD163 positive events were compensated for unstained events by subtraction. A 5% error margin was accepted in the unstained image. GLN and IL-1/LPS showed no interaction on the percentage of CD163+ events (3b). The percentage of CD163+ events was significantly increased by GLN but not affected by IL-1/LPS. GLN and IL-1/LPS significantly interacted on the number of CD163+ macrophages (3c). CD163+ macrophage numbers were significantly increased in GLN+IL-1/LPS+ rats by both GLN and IL-1/LPS. Fig 3d shows immunofluorescence representation of CD163 stained lung regions from the GLN-IL-1/LPS+ and GLN+IL-1/LPS+ groups (white arrow points to zoomed cell in bottom right corner) (DAPI nuclear staining = blue; CD163 = red). (The significance of differences among the four groups was analyzed by two-way ANOVA. See statistical methods section for details on paired comparisons. Statistical significance was accepted as p<0.05: * compared to respective GLN- group, # compared to respective IL-1/LPS- group, ^ significant interaction between GLN and IL-1/LPS group).
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pone.0130764.g003: Quantification of CD163+ BAL cells by flow-cytometry.Fig (3a) shows representative flow-cytometry histograms from studied groups. Fluorescence was compared to unstained controls for 35,000 events in each histogram display, and the stained APC-CD163 positive events were compensated for unstained events by subtraction. A 5% error margin was accepted in the unstained image. GLN and IL-1/LPS showed no interaction on the percentage of CD163+ events (3b). The percentage of CD163+ events was significantly increased by GLN but not affected by IL-1/LPS. GLN and IL-1/LPS significantly interacted on the number of CD163+ macrophages (3c). CD163+ macrophage numbers were significantly increased in GLN+IL-1/LPS+ rats by both GLN and IL-1/LPS. Fig 3d shows immunofluorescence representation of CD163 stained lung regions from the GLN-IL-1/LPS+ and GLN+IL-1/LPS+ groups (white arrow points to zoomed cell in bottom right corner) (DAPI nuclear staining = blue; CD163 = red). (The significance of differences among the four groups was analyzed by two-way ANOVA. See statistical methods section for details on paired comparisons. Statistical significance was accepted as p<0.05: * compared to respective GLN- group, # compared to respective IL-1/LPS- group, ^ significant interaction between GLN and IL-1/LPS group).

Mentions: Fig 3a depicts a representative flow-cytometry histogram of each of the 4 groups. No significant interaction was found between GLN pretreatment and IL-1/LPS insufflation on the percentage of CD163+ macrophages in the reconstituted BAL cell pellet. GLN pretreatment significantly increased the percentage of CD163+ macrophages without any significant effect induced by IL-1/LPS (Fig 3b). This percentage of CD163+ macrophages was multiplied by the total BAL macrophage count to calculate the CD163+ macrophage counts for each rat (Fig 3c). Both GLN and IL-1/LPS significantly interacted on the observed increased CD163+ macrophage counts in the GLN-pretreated IL-1/LPS-insufflated group compared to the other three groups. Immunofluorescence located CD163 staining in alveolar macrophages (Fig 3d).


Brief Glutamine Pretreatment Increases Alveolar Macrophage CD163/Heme Oxygenase-1/p38-MAPK Dephosphorylation Pathway and Decreases Capillary Damage but Not Neutrophil Recruitment in IL-1/LPS-Insufflated Rats.

Fernandez-Bustamante A, Agazio A, Wilson P, Elkins N, Domaleski L, He Q, Baer KA, Moss AF, Wischmeyer PE, Repine JE - PLoS ONE (2015)

Quantification of CD163+ BAL cells by flow-cytometry.Fig (3a) shows representative flow-cytometry histograms from studied groups. Fluorescence was compared to unstained controls for 35,000 events in each histogram display, and the stained APC-CD163 positive events were compensated for unstained events by subtraction. A 5% error margin was accepted in the unstained image. GLN and IL-1/LPS showed no interaction on the percentage of CD163+ events (3b). The percentage of CD163+ events was significantly increased by GLN but not affected by IL-1/LPS. GLN and IL-1/LPS significantly interacted on the number of CD163+ macrophages (3c). CD163+ macrophage numbers were significantly increased in GLN+IL-1/LPS+ rats by both GLN and IL-1/LPS. Fig 3d shows immunofluorescence representation of CD163 stained lung regions from the GLN-IL-1/LPS+ and GLN+IL-1/LPS+ groups (white arrow points to zoomed cell in bottom right corner) (DAPI nuclear staining = blue; CD163 = red). (The significance of differences among the four groups was analyzed by two-way ANOVA. See statistical methods section for details on paired comparisons. Statistical significance was accepted as p<0.05: * compared to respective GLN- group, # compared to respective IL-1/LPS- group, ^ significant interaction between GLN and IL-1/LPS group).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493112&req=5

pone.0130764.g003: Quantification of CD163+ BAL cells by flow-cytometry.Fig (3a) shows representative flow-cytometry histograms from studied groups. Fluorescence was compared to unstained controls for 35,000 events in each histogram display, and the stained APC-CD163 positive events were compensated for unstained events by subtraction. A 5% error margin was accepted in the unstained image. GLN and IL-1/LPS showed no interaction on the percentage of CD163+ events (3b). The percentage of CD163+ events was significantly increased by GLN but not affected by IL-1/LPS. GLN and IL-1/LPS significantly interacted on the number of CD163+ macrophages (3c). CD163+ macrophage numbers were significantly increased in GLN+IL-1/LPS+ rats by both GLN and IL-1/LPS. Fig 3d shows immunofluorescence representation of CD163 stained lung regions from the GLN-IL-1/LPS+ and GLN+IL-1/LPS+ groups (white arrow points to zoomed cell in bottom right corner) (DAPI nuclear staining = blue; CD163 = red). (The significance of differences among the four groups was analyzed by two-way ANOVA. See statistical methods section for details on paired comparisons. Statistical significance was accepted as p<0.05: * compared to respective GLN- group, # compared to respective IL-1/LPS- group, ^ significant interaction between GLN and IL-1/LPS group).
Mentions: Fig 3a depicts a representative flow-cytometry histogram of each of the 4 groups. No significant interaction was found between GLN pretreatment and IL-1/LPS insufflation on the percentage of CD163+ macrophages in the reconstituted BAL cell pellet. GLN pretreatment significantly increased the percentage of CD163+ macrophages without any significant effect induced by IL-1/LPS (Fig 3b). This percentage of CD163+ macrophages was multiplied by the total BAL macrophage count to calculate the CD163+ macrophage counts for each rat (Fig 3c). Both GLN and IL-1/LPS significantly interacted on the observed increased CD163+ macrophage counts in the GLN-pretreated IL-1/LPS-insufflated group compared to the other three groups. Immunofluorescence located CD163 staining in alveolar macrophages (Fig 3d).

Bottom Line: Glutamine (GLN) attenuates acute lung injury (ALI) but its effect on alveolar macrophages is unknown.GLN pretreatment before IL-1/LPS also significantly increased HO-1 concentrations and dephosphorylated p38-MAPK levels but not cytokine levels in alveolar macrophages.Short-term GLN pretreatment activates the anti-inflammatory CD163/HO-1/p38-MAPK dephosphorylation pathway of alveolar macrophages and decreases capillary damage but not neutrophil recruitment in IL-1/LPS-insufflated rats.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, University of Colorado SOM, Aurora, Colorado, United States of America; Webb-Waring Center, University of Colorado SOM, Aurora, Colorado, United States of America.

ABSTRACT

Background: Glutamine (GLN) attenuates acute lung injury (ALI) but its effect on alveolar macrophages is unknown. We hypothesized that GLN pretreatment would induce the anti-inflammatory CD163/heme oxygenase (HO)-1/p38-MAPK dephosphorylation pathway in alveolar macrophages and reduce ALI in rats insufflated with interleukin-1 (IL-1) and lipopolysaccharide (LPS).

Methods: Male Sprague-Dawley rats were randomized to the following groups: GLN-IL-1/LPS-, GLN+IL-1/LPS-, GLN-IL-1/LPS+, and GLN+IL-1/LPS+. GLN pretreatment was given via gavage (1 g/kg L-alanyl-L-glutamine) daily for 2 days. ALI was subsequently induced by insufflating 50 ng IL-1 followed by 5mg/kg E.coli LPS. After 24h, bronchoalveolar lavage (BAL) protein, lactate dehydrogenase (LDH) and neutrophil concentrations were analyzed. BAL alveolar macrophage CD163+ expression, HO-1 and p38-MAPK concentrations were measured, as well as alveolar macrophage tumor necrosis factor (TNF)-α and interleukin (IL)-10 concentrations. Histology and immunofluorescence studies were also performed.

Results: Following IL-1/LPS insufflation, GLN pretreated rats had significantly decreased BAL protein and LDH concentrations, but not BAL neutrophil counts, compared to non-GLN pretreated rats. The number of alveolar macrophages and the number of CD163+ macrophages were significantly increased in GLN pretreated IL-1/LPS-insufflated rats compared to non-GLN pretreated, IL-1/LPS-insufflated rats. GLN pretreatment before IL-1/LPS also significantly increased HO-1 concentrations and dephosphorylated p38-MAPK levels but not cytokine levels in alveolar macrophages. Immunofluorescence localized CD163 and HO-1 in alveolar macrophages.

Conclusion: Short-term GLN pretreatment activates the anti-inflammatory CD163/HO-1/p38-MAPK dephosphorylation pathway of alveolar macrophages and decreases capillary damage but not neutrophil recruitment in IL-1/LPS-insufflated rats.

No MeSH data available.


Related in: MedlinePlus