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. 2014 Apr;76(4):982-90.
doi: 10.1097/TA.0000000000000163.

Alveolar macrophage depletion increases the severity of acute inflammation following nonlethal unilateral lung contusion in mice

David Machado-Aranda  1 , Madathilparambil V Suresh, Bi Yu, Vladislov Dolgachev, Mark R Hemmila, Krishnan Raghavendran
Affiliations

Alveolar macrophage depletion increases the severity of acute inflammation following nonlethal unilateral lung contusion in mice

David Machado-Aranda et al. J Trauma Acute Care Surg. 2014 Apr.

Abstract

Background: Lung contusion (LC) is a common injury resulting from blunt thoracic trauma. LC is an important risk factor for the development acute lung injury, adult respiratory distress syndrome, and ventilator-associated pneumonia, all of which increase mortality from trauma. LC produces a nonspecific immune cellular response. Neutrophil recruitment is known to increase the severity of inflammation during LC. However, the exact role of macrophages in modulating the response to LC has not been well described.

Methods: We used a cortical contusion impactor to induce unilateral LC in mice. Thoracic micro computed tomographic scans of these animals were obtained to document radiologic changes over time following LC. To understand the role of macrophages during LC, liposomal clodronate was used to deplete macrophage levels before traumatic insult. Acute inflammatory attributes after LC were assessed, by measuring pressure-volume mechanics; quantifying bronchial alveolar lavage levels of leukocytes, albumin, and cytokines; and finally examining lung specimen histopathology at 5, 24, 48, and 72 hours after injury.

Results: After LC, alveolar macrophage numbers were significantly reduced and exhibited slowed recovery. Simultaneously, there was a significant increase in bronchial alveolar lavage neutrophil counts. The loss of macrophages could be attributed to both cellular apoptosis and necrosis. Pretreatment with clodronate increased the severity of lung inflammation as measured by worsened pulmonary compliance, increased lung permeability, amplification of neutrophil recruitment, and increases in early proinflammatory cytokine levels.

Conclusion: The presence of regulatory alveolar macrophages plays an important role in the pathogenesis of acute inflammation following LC.

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Figures

Figure 1
Figure 1
Evolution of unilateral LC injury in mice. Images were captured in nongated micro CT scanner with midthoracic axial cuts. Infiltrates (white arrow) can be seen as early as 10 minutes and are most severe between 24 hours and 48 hours. If no additional insult was added, lung infiltrates will show almost complete resolution by 7 days.
Figure 2
Figure 2
Macrophages are lost during LC injury as a result of increased necrosis and apoptosis. A, BAL cells were counted and later manually differentiated at different time points following LC. Macrophages showed a persistent reduction in numbers when compared with uninjured controls at all time points, whereas neutrophils showed up to 20-fold increases as compared with baseline. Dendritic cells had a modest increased that was not statically significant. B, BAL cells were analyzed using flow cytometry. Necrosis as characterized by Live-Dead+/Annexin V− double stain was present in all time points assessed in F480+/Gr− surface markers present in activated macrophages. Early-stage (Live-Dead−/Annexin V+) and late-stage (Live-Dead+/Annexin V+) apoptoses were also present but mostly so at 48 hours and 72 hours. One-way ANOVA versus uninjured control (n = 9, *p < 0.05. **p < 0.01. ***p < 0.001).
Figure 3
Figure 3
Clodronate pretreatment decreased volumes for pressure changes in mechanically ventilated mice following LC. Uninjured controls are shown in open triangles, Liposome-pretreated animals are shown in solid triangles and clodronate-pretreated animals are in solid boxes. “Quasi-static” closed-chest P/V curves were measured at 5, 24, 48, and 72 hours after injury. Each curve was generated by more than 4,000 data points for each animal, and SEM for nine animals is not shown to allow clear visualization of the curves.
Figure 4
Figure 4
Clodronate pretreatment worsens permeability injury in mice with LC. Mice receiving clodronate pretreatment (100 μL of 0.2 mg/mL) IP injection 48 hours before LC showed increase alveolar/endothelial permeability to albumin recovered in BAL. These effects were mostly noted at 5-hour and 24-hour peak closely following other inflammatory attributes (P/V curves, BAL cellularity). One-way ANOVA versus uninjured control (n = 9, *p < 0.05. **p < 0.01. ***p < 0.001). One-way ANOVA versus liposome (n = 9, ##p < 0.01).
Figure 5
Figure 5
Effect on BAL cellular composition with macrophage-depleting agent clodronate in the evolution of LC injury. A, An additional group of animals were sacrificed 48 hours after pretreatment injection; values show the composition of BAL before LC. B, Evolution of the total number of cells present in BAL after LC. Clodronate treatment decreased the total number of cells present in BAL at all compared time points. C, With the use of F4/80+/CD11b+ antibody labeling, macrophage numbers were measured by flow cytometry. Decreases in total number of cells were attributed overall to diminished number of total macrophages after clodronate treatment. D, Neutrophils measured by flow cytometry using Gr-1+/CD11b+/F4/80− labeling showed that within the clodronate group, there was a significant, more than 100-fold increase of neutrophilic at 5 hours. Neutrophilia was persistent at all time points. One-way ANOVA versus uninjured control (n = 9, *p < 0.05. **p < 0.01. ***p < 0.001). One-way ANOVA versus liposome (n = 9, ##p < 0.01. ###p < 0.001).
Figure 6
Figure 6
Evolution of histologic changes in the presence of macrophage-depleting agent clodronate following LC injury. The right lungs at the area of grossly identified contusion were fixed and processed for hematoxylin and eosin staining. Clodronate groups showed increase inflammatory attributes (hemorrhage, cellular infiltrates, alveolar edema) in comparison with respective controls at early time points (5, 24, and 48 hours). At 72 hours, both groups (liposome and clodronate) seem to have similar recovery from LC injury. See text for details. Bar, 50 μm.
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