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Efficacy and safety of inhaled carbon monoxide during pulmonary inflammation in mice.

Wilson MR, O'Dea KP, Dorr AD, Yamamoto H, Goddard ME, Takata M - PLoS ONE (2010)

Bottom Line: Here we investigate the efficacy, safety and mechanism of action of low dose inhaled carbon monoxide (CO) using a mouse model of lipopolysaccharide (LPS)-induced pulmonary inflammation.In contrast to such apparently beneficial effects, 100 ppm inhaled CO induced an increase in pulmonary barrier permeability as determined by lavage fluid protein content and translocation of labelled albumin from blood to the alveolar space.Overall, these data confirm some protective role for inhaled CO during pulmonary inflammation, although this required a dose that produced carboxyhemoglobin values close to potentially toxic levels for humans, and increased lung permeability.

View Article: PubMed Central - PubMed

Affiliation: Anaesthetics, Pain Medicine and Intensive Care, Imperial College London, Chelsea and Westminster Hospital, London, UK. michael.wilson@imperial.ac.uk

ABSTRACT

Background: Pulmonary inflammation is a major contributor to morbidity in a variety of respiratory disorders, but treatment options are limited. Here we investigate the efficacy, safety and mechanism of action of low dose inhaled carbon monoxide (CO) using a mouse model of lipopolysaccharide (LPS)-induced pulmonary inflammation.

Methodology: Mice were exposed to 0-500 ppm inhaled CO for periods of up to 24 hours prior to and following intratracheal instillation of 10 ng LPS. Animals were sacrificed and assessed for intraalveolar neutrophil influx and cytokine levels, flow cytometric determination of neutrophil number and activation in blood, lung and lavage fluid samples, or neutrophil mobilisation from bone marrow.

Principal findings: When administered for 24 hours both before and after LPS, inhaled CO of 100 ppm or more reduced intraalveolar neutrophil infiltration by 40-50%, although doses above 100 ppm were associated with either high carboxyhemoglobin, weight loss or reduced physical activity. This anti-inflammatory effect of CO did not require pre-exposure before induction of injury. 100 ppm CO exposure attenuated neutrophil sequestration within the pulmonary vasculature as well as LPS-induced neutrophilia at 6 hours after LPS, likely due to abrogation of neutrophil mobilisation from bone marrow. In contrast to such apparently beneficial effects, 100 ppm inhaled CO induced an increase in pulmonary barrier permeability as determined by lavage fluid protein content and translocation of labelled albumin from blood to the alveolar space.

Conclusions: Overall, these data confirm some protective role for inhaled CO during pulmonary inflammation, although this required a dose that produced carboxyhemoglobin values close to potentially toxic levels for humans, and increased lung permeability.

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Tissue neutrophil numbers 6 hours after LPS, determined by flow cytometry.Neutrophil (PMN) number in lavage fluid (A), lung tissue (B) and blood (C) from untreated mice (no LPS or CO), or mice exposed to 0 or 100 ppm carbon monoxide (CO) for 6 hours after LPS instillation. Single cell suspensions were prepared from excised lungs of mice by mechanical disruption. Lavage, lung and blood cell samples were stained with fluorochrome-conjugated antibodies against cell-surface markers (CD11b, F4/80, Gr-1, L-selectin) and analysed by flow cytometry. Microsphere counting beads were added to enable cell quantification. Neutrophils were identified based on forward/side-scatter properties and F4/80 and Gr-1 expression. *p<0.05, **p<0.01 vs LPS +0 ppm CO; n = 9–10/group.
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pone-0011565-g006: Tissue neutrophil numbers 6 hours after LPS, determined by flow cytometry.Neutrophil (PMN) number in lavage fluid (A), lung tissue (B) and blood (C) from untreated mice (no LPS or CO), or mice exposed to 0 or 100 ppm carbon monoxide (CO) for 6 hours after LPS instillation. Single cell suspensions were prepared from excised lungs of mice by mechanical disruption. Lavage, lung and blood cell samples were stained with fluorochrome-conjugated antibodies against cell-surface markers (CD11b, F4/80, Gr-1, L-selectin) and analysed by flow cytometry. Microsphere counting beads were added to enable cell quantification. Neutrophils were identified based on forward/side-scatter properties and F4/80 and Gr-1 expression. *p<0.05, **p<0.01 vs LPS +0 ppm CO; n = 9–10/group.

Mentions: As the attenuation of alveolar neutrophilia was seemingly not associated with changes in soluble mediator levels, we investigated whether CO influenced early neutrophil activation and sequestration within the lung microvasculature, which has been shown to peak between 4–12 hours following intratracheal LPS [20]. This was assessed using flow cytometry to determine neutrophil numbers and adhesion molecule expression in blood, lung tissue and lavage fluid. 6 hours of LPS treatment induced a significant increase in neutrophil numbers in each of these compartments. 100 ppm CO tended to reduce neutrophil numbers in lavage fluid, although this was not significant at this early point (fig. 6A). Neutrophil sequestration within the lung tissue was however significantly attenuated by CO (fig. 6B). Similarly, the increase in blood neutrophils following LPS was almost completely abrogated by CO (fig. 6C). To determine whether this latter finding was related to an effect of CO to inhibit neutrophil mobilisation from bone marrow (as opposed to other ‘marginated’ pools), mice were dosed with BrdU 48 hours before LPS administration. LPS induced a clear increase in the number of BrdU-containing (i.e. newly released from bone marrow) neutrophils in the blood at 6 hours, which was significantly attenuated by 100 ppm CO exposure (fig. 7). A similar pattern was observed in the lung although changes were not significant.


Efficacy and safety of inhaled carbon monoxide during pulmonary inflammation in mice.

Wilson MR, O'Dea KP, Dorr AD, Yamamoto H, Goddard ME, Takata M - PLoS ONE (2010)

Tissue neutrophil numbers 6 hours after LPS, determined by flow cytometry.Neutrophil (PMN) number in lavage fluid (A), lung tissue (B) and blood (C) from untreated mice (no LPS or CO), or mice exposed to 0 or 100 ppm carbon monoxide (CO) for 6 hours after LPS instillation. Single cell suspensions were prepared from excised lungs of mice by mechanical disruption. Lavage, lung and blood cell samples were stained with fluorochrome-conjugated antibodies against cell-surface markers (CD11b, F4/80, Gr-1, L-selectin) and analysed by flow cytometry. Microsphere counting beads were added to enable cell quantification. Neutrophils were identified based on forward/side-scatter properties and F4/80 and Gr-1 expression. *p<0.05, **p<0.01 vs LPS +0 ppm CO; n = 9–10/group.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2903490&req=5

pone-0011565-g006: Tissue neutrophil numbers 6 hours after LPS, determined by flow cytometry.Neutrophil (PMN) number in lavage fluid (A), lung tissue (B) and blood (C) from untreated mice (no LPS or CO), or mice exposed to 0 or 100 ppm carbon monoxide (CO) for 6 hours after LPS instillation. Single cell suspensions were prepared from excised lungs of mice by mechanical disruption. Lavage, lung and blood cell samples were stained with fluorochrome-conjugated antibodies against cell-surface markers (CD11b, F4/80, Gr-1, L-selectin) and analysed by flow cytometry. Microsphere counting beads were added to enable cell quantification. Neutrophils were identified based on forward/side-scatter properties and F4/80 and Gr-1 expression. *p<0.05, **p<0.01 vs LPS +0 ppm CO; n = 9–10/group.
Mentions: As the attenuation of alveolar neutrophilia was seemingly not associated with changes in soluble mediator levels, we investigated whether CO influenced early neutrophil activation and sequestration within the lung microvasculature, which has been shown to peak between 4–12 hours following intratracheal LPS [20]. This was assessed using flow cytometry to determine neutrophil numbers and adhesion molecule expression in blood, lung tissue and lavage fluid. 6 hours of LPS treatment induced a significant increase in neutrophil numbers in each of these compartments. 100 ppm CO tended to reduce neutrophil numbers in lavage fluid, although this was not significant at this early point (fig. 6A). Neutrophil sequestration within the lung tissue was however significantly attenuated by CO (fig. 6B). Similarly, the increase in blood neutrophils following LPS was almost completely abrogated by CO (fig. 6C). To determine whether this latter finding was related to an effect of CO to inhibit neutrophil mobilisation from bone marrow (as opposed to other ‘marginated’ pools), mice were dosed with BrdU 48 hours before LPS administration. LPS induced a clear increase in the number of BrdU-containing (i.e. newly released from bone marrow) neutrophils in the blood at 6 hours, which was significantly attenuated by 100 ppm CO exposure (fig. 7). A similar pattern was observed in the lung although changes were not significant.

Bottom Line: Here we investigate the efficacy, safety and mechanism of action of low dose inhaled carbon monoxide (CO) using a mouse model of lipopolysaccharide (LPS)-induced pulmonary inflammation.In contrast to such apparently beneficial effects, 100 ppm inhaled CO induced an increase in pulmonary barrier permeability as determined by lavage fluid protein content and translocation of labelled albumin from blood to the alveolar space.Overall, these data confirm some protective role for inhaled CO during pulmonary inflammation, although this required a dose that produced carboxyhemoglobin values close to potentially toxic levels for humans, and increased lung permeability.

View Article: PubMed Central - PubMed

Affiliation: Anaesthetics, Pain Medicine and Intensive Care, Imperial College London, Chelsea and Westminster Hospital, London, UK. michael.wilson@imperial.ac.uk

ABSTRACT

Background: Pulmonary inflammation is a major contributor to morbidity in a variety of respiratory disorders, but treatment options are limited. Here we investigate the efficacy, safety and mechanism of action of low dose inhaled carbon monoxide (CO) using a mouse model of lipopolysaccharide (LPS)-induced pulmonary inflammation.

Methodology: Mice were exposed to 0-500 ppm inhaled CO for periods of up to 24 hours prior to and following intratracheal instillation of 10 ng LPS. Animals were sacrificed and assessed for intraalveolar neutrophil influx and cytokine levels, flow cytometric determination of neutrophil number and activation in blood, lung and lavage fluid samples, or neutrophil mobilisation from bone marrow.

Principal findings: When administered for 24 hours both before and after LPS, inhaled CO of 100 ppm or more reduced intraalveolar neutrophil infiltration by 40-50%, although doses above 100 ppm were associated with either high carboxyhemoglobin, weight loss or reduced physical activity. This anti-inflammatory effect of CO did not require pre-exposure before induction of injury. 100 ppm CO exposure attenuated neutrophil sequestration within the pulmonary vasculature as well as LPS-induced neutrophilia at 6 hours after LPS, likely due to abrogation of neutrophil mobilisation from bone marrow. In contrast to such apparently beneficial effects, 100 ppm inhaled CO induced an increase in pulmonary barrier permeability as determined by lavage fluid protein content and translocation of labelled albumin from blood to the alveolar space.

Conclusions: Overall, these data confirm some protective role for inhaled CO during pulmonary inflammation, although this required a dose that produced carboxyhemoglobin values close to potentially toxic levels for humans, and increased lung permeability.

Show MeSH
Related in: MedlinePlus