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

<div><p>Background</p><p>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).</p><p>Methods</p><p>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 (1g/kg L-alanyl-L-glutamine) daily for 2 days. ALI was subsequently induced by insufflating 50ng IL-1 followed by 5mg/kg <i>E</i>.<i>coli</i> 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.</p><p>Results</p><p>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.</p><p>Conclusion</p><p>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.</p></div

Measurements of HO-1 and dephosphorylated p38-MAPK concentrations in alveolar macrophages.

<p>The concentration of HO-1 in isolated alveolar macrophages (<b>4a</b>) was significantly increased exclusively by IL-1/LPS with no interaction or effect of GLN pretreatment. Fig <b>4b</b> shows immunofluorescence representation of HO-1 stained lung regions from GLN-IL-1/LPS+ and GLN+IL-1/LPS+ groups. (white arrow points to zoomed cell in bottom right corner) (DAPI nuclear staining = blue; HO-1 = pink). Dephosphorylated p38-MAPK concentrations (<b>4c</b>) were significantly increased in the GLN+IL-1/LPS+ group compared to the other three groups, and both GLN and IL-1/LPS had a significantly interaction on this increase. (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).</p

Quantification of CD163+ BAL cells by flow-cytometry.

<p>Fig (<b>3a</b>) 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 (<b>3b</b>). 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 (<b>3c</b>). CD163+ macrophage numbers were significantly increased in GLN+IL-1/LPS+ rats by both GLN and IL-1/LPS. Fig <b>3d</b> 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).</p

Lung injury features.

<p>There was no interaction between GLN pretreatment and IL-1/LPS insufflation on BAL protein concentrations (1a). IL-1/LPS significantly increased BAL protein levels in both GLN+ and GLN- groups. GLN+IL-1/LPS+ groups had significantly lower BAL protein concentrations than GLN-IL-1/LPS+ groups. GLN and IL-1/LPS showed a significant interaction on BAL LDH concentration (1b). BAL LDH concentrations were significantly higher in GLN-IL-1/LPS+ groups compared to both IL-1/LPS- groups. GLN+IL-1/LPS+ rats had significantly lower BAL LDH levels than GLN-IL-1/LPS+ groups. Fig 1c shows representative H&E histology images from each group. IL-1/LPS insufflation increased the blindly assessed histological lung injury scores with no interaction or effect by GLN pretreatment (1d). (The significance of differences among the four groups was analyzed by two-way ANOVA followed by paired comparisons. Statistical significance was accepted as p<0.05: * compared to GLN-IL-1/LPS- group, # compared to respective GLN+IL-1/LPS- group, ^ compared to GLN-IL-1/LPS+ group).</p

Total BAL cell numbers and cell differential counts.

<p>IL-1/LPS significantly increased the BAL total cell numbers without a significant effect by GLN pretreatment (2a). No interaction between GLN and IL-1/LPS was observed on BAL neutrophil numbers (2b). BAL neutrophils significantly increased with IL-1/LPS insufflation but were not significantly affected by GLN pretreatment. Conversely, GLN and IL-1/LPS showed a significant interaction on BAL macrophage numbers (2c) with both GLN and IL-1/LPS significantly increasing the numbers of BAL macrophages in the GLN+IL-1/LPS+ group compared to the other three groups. (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 GLN-IL-1/LPS- group, # compared to respective GLN+IL-1/LPS- group, ^ compared to GLN-IL-1/LPS+ group).</p