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Am J Physiol Lung Cell Mol Physiol 294: L644-L653, 2008. First published January 18, 2008; doi:10.1152/ajplung.00324.2007
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EDITORIAL FOCUS

Low-dose carbon monoxide treatment attenuates early pulmonary neutrophil recruitment after acid aspiration

Jean A. Nemzek,1,2 Christopher Fry,1 and Omorodola Abatan1

1Unit for Laboratory Animal Medicine and 2Department of Pathology, University of Michigan, Ann Arbor, Michigan

Submitted 14 August 2007 ; accepted in final form 11 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Exogenous carbon monoxide (CO) has anti-inflammatory and cytoprotective properties that show promise in the treatment of numerous pulmonary diseases. However, the effectiveness of CO in acute pulmonary injury associated with direct lung insult has not been shown conclusively. The purpose of this study was to determine if exogenous CO would modulate the pulmonary inflammation and lung injury that develops after acid aspiration. Groups of mice were given intratracheal (IT) injections of either saline or an acidic solution. After the IT injection, some of the mice in each group were allowed to spontaneously inhale CO (500 ppm). Mice exposed to CO for 6 h after IT acid had a significant decrease in bronchoalveolar lavage (BAL) fluid neutrophil counts and in histological evidence of lung injury. These results could not be explained by changes in BAL fluid chemokine levels or altered CXCR2 expression. The reduced neutrophil recruitment was associated with a decrease in the percentage of peripheral blood neutrophils expressing CD11b protein. However, within 24 h, the BAL neutrophil counts increased and were not different from animals without CO exposure. In addition, indices of vascular integrity were not different between animals with acid aspiration regardless of CO exposure at the later time point. These results showed that CO can modulate the early development of acute lung inflammation in this model of acid aspiration. Although these effects were eventually overwhelmed, the results suggest that CO may have efficacy during the initial treatment of aspiration lung injury.

lung injury; chemokines; CXCR2; CD11b; inflammation

ASPIRATION OF GASTRIC ACID is a leading cause of pulmonary complications in patients with neurological or anatomical compromise of reflexes protecting the airway. Experimental studies have shown that aspiration-associated lung pathology develops in a biphasic pattern (16). Initially, chemical irritation produces an increase in vascular permeability without substantial inflammation (16). The second phase, beginning within 2–3 h of aspiration, is an innate immune response (16) characterized by dramatic increases of local proinflammatory cytokines (TNF, IL-6, chemokines) and neutrophil recruitment to the lung (8, 13, 15). The accumulation of neutrophils occurs not only at sites directly affected by the acidic aspirate but also into pulmonary sites remote from the acid injury (11, 19). These neutrophils are key mediators of a progressive inflammatory response, and acid aspiration lung injury is greatly reduced in neutropenic animals (11). Similarly, lung injury may be reduced by neutralization of adhesion molecules (21) or chemoattractants (8, 33) specific for neutrophils. In particular, IL-8 (8) and its rodent homologs, MIP-2{alpha} and KC (CINC) (23, 33), have been linked to pulmonary neutrophil recruitment after aspiration. Experimental studies have shown that specific chemokine levels can predict the severity of the aspiration insult and the progression of the lung injury (13). After neutrophils are recruited into the lung and airways, proteases and oxygen radicals contribute to organ damage (17, 20), leading to loss of vascular integrity and edema. The lung injury generally peaks within 6–8 h of the insult (17, 23) and may resolve uneventfully. However, aspiration lung injury may progress to more severe sequelae including fulminate acute respiratory distress syndrome and increased susceptibility to bacterial pneumonia (18). The serious morbidity and mortality that can accompany the acute lung injury highlight the need for effective treatment of acid aspiration.

Recently, compelling studies have shown that inducible heme oxygenase-1 (HO-1) and a byproduct of heme metabolism, carbon monoxide (CO), modulate a number of disease processes (31, 34). In addition, experimental studies have shown that non-toxic, low doses of CO (250–500 ppm) provide anti-inflammatory and cytoprotective effects in specific models of pulmonary disease (4, 9, 29, 36). The production of proinflammatory cytokines was reduced by CO exposure in models of allergic (4), ventilator-induced (7), and hyperoxic lung injury (29). In addition, exposure to CO reduced inflammatory cell recruitment to the lung and airways after pulmonary insult (4, 28, 29, 36). These apparently beneficial results have primarily been documented in models of lung injury involving exposure to an insult over several hours to days. When a more severe, hyperacute injury was addressed in one study, CO exposure had no effect on the lung inflammation induced within four hours of either LPS or oleic acid administration (10). The contrasting results from these models suggest that additional studies are needed to validate the effectiveness of exogenous CO therapy in acute lung injury.

Exogenous CO might prove beneficial in acid aspiration lung injury by attenuating proinflammatory cytokine production and neutrophil recruitment. However, the progression and time course of the lung injury induced by aspiration are not well represented by the models previously used in experimental studies evaluating CO. Therefore, the purpose of this study was to examine the effects of low-dose, exogenous CO (500 ppm) on lung inflammation and injury in a murine model of acid aspiration. In addition, we examined several factors that could contribute to the effects of CO on neutrophil recruitment to the lung including chemokine levels, chemokine receptor expression, peripheral blood neutrophil counts, and surface expression of CD11b on neutrophils. The results demonstrated an acute reduction in neutrophil recruitment to the lung that appears to be mediated by exposure to CO even without pretreatment. Although the acute lung injury appeared to be progressive in spite of continuous treatment, these studies provide a basis for further investigations of CO treatment after acid aspiration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Study design. To induce acid aspiration lung injury, mice were given intratracheal (IT) injections of either an acidic solution or saline as a control. In each lung injury group, one-half of the mice received treatment with CO; the other did not. Treatment with CO was continuous from the time of IT injection to the time of euthanasia (6 or 24 h post-IT injection) unless otherwise noted. Blood, bronchoalveolar lavage (BAL) fluid, and lung tissue were collected to examine lung inflammation and injury. Peripheral neutrophils were examined for factors that could affect their recruitment to the lung including absolute counts and markers of activation.

Animals. Female ICR mice (23–25 g) were obtained from Harlan Sprague Dawley (Indianapolis, IN). The mice were housed in a temperature-controlled room with a 12-h dark/light cycle. Food and water were given ad libitum. The University Committee on Use and Care of Animals approved all of the experiments.

Aspiration. Mice were anesthetized with isoflurane, and a total of 80 µl of solution was delivered IT using methods previously described (24). The acidic solution consisted of saline titrated to a pH of 1.15 with hydrochloric acid.

CO treatment. An atmospheric chamber linked to a CO mixing chamber was used for CO exposure. Gas was supplied via a pressurized gas cylinder and mixed with room air by an inline blower regulated by a reostat. The system was set to deliver 500 ppm CO. The CO level was measured by a sensor (SEC2000) linked to a data logger (VERITEQ sp-4000) and to a computer (Pioneer Gas Monitor) that regulated the gas flow valve (Alicat series 16, Alicat Scientific). The system was professionally calibrated (Argus Group, Chesterfield, MI) before initiating the study. Before each experiment, the system was allowed to equilibrate for one-half hour before animals were placed in the chamber. During actual delivery, the CO levels fluctuated from 485–510 ppm.

Sample harvest. The mice were anesthetized with intraperitoneal injections of 87 mg/kg ketamine (Ketaset; Fort Dodge Laboratories, Fort Dodge, IA) and 13 mg/kg xylazine (Rompun; Bayer, Shawnee Mission, KS). A 20-µl sample of blood was collected in EDTA for a complete blood count and then mice were exsanguinated by the retroorbital route. After blood collection, the anesthetized animals were killed by cervical dislocation. A BAL was performed by injecting two separate 1-ml volumes of warm Hanks' balanced salt solution (HBSS without Ca2Cl, Mg2S04, or phenol red; GIBCO, Grand Island, NJ) into the trachea. The right ventricle was then perfused with 2 ml of saline. After BAL and perfusion, the left lung lobe from each mouse was placed in 10% buffered formalin for histological analysis.

Lung tissue digestion. After BAL and heart perfusion, lungs were excised. Lungs were dispersed in collagenase type 3 (Worthington Biochemical, Lakewood, NJ) in HBSS with fetal bovine serum (Invitrogen, Carlsbad, CA) and deoxyribonuclease I (Worthington Biochemical) at 37°C for 45 min. Samples were filtered (100 µm) before processing for flow cytometry.

Peripheral blood analysis. A complete blood count was performed using a Hemavet Mascot Multispecies Hematology System Counter (CDC Technologies, Oxford, CT). The remaining blood was centrifuged at (2,000 g, 5 min), and plasma was stored (–20°C) for later cytokine analysis.

BAL cell counts and differential. The 1-ml samples were centrifuged (600 g, 5 min), and the supernatant from the first sample retrieved from each mouse was stored at –20°C. The cell pellets from the two samples were pooled for total counts with a Coulter Counter model Z1 after red blood cell lysis with Zap-Oglobin II (Coulter, Miami, FL). Slides were loaded with 1 x 105 cells, centrifuged (700 g, 5 min), and stained with Diff-Quick (Baxter, Detroit, MI). Differentials (300 cells) were counted with a light microscope.

Histology. The lung samples were fixed in formalin, embedded, sectioned, and stained (hematoxylin and eosin). Sections were evaluated under light microscopy by a blinded observer using a previously described scoring system (2, 26). Scores (0–4) were assigned for alveolar congestion, hemorrhage, infiltration of neutrophils, and alveolar wall thickness. Cumulative numbers for each lung were used to determine the mean and median scores for each group.

Albumin ELISA. Albumin levels were measured in BAL fluid using a direct ELISA as previously described (23). A standard curve of mouse albumin (Sigma) and lung lavage samples (1:100) were diluted in a borate buffer (120 mM NaCl, 50 mM H3BO4, 16 N NaOH). A polyclonal rabbit anti-mouse albumin antibody (6.9 µg/ml) was diluted 1:8,000 in a buffer consisting of PBS with 10% Blocker Casein in PBS (Pierce, Rockford, IL) and used as the primary antibody. The secondary antibody was goat anti-rabbit IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA) and was also diluted 1:8,000. The color reagent was 3,3',5,5'-tetramethylbenzidine (TMB), and the reaction was stopped with 1.5 N sulfuric acid. The absorbance was read on an ELISA plate reader (Bio-Tek Instruments, Windoski, VT) at 450 nm and 630 nm.

Lung wet-to-dry weight ratio. In separate groups of animals, lung lobes were removed immediately after euthanasia without performing BAL or heart perfusion. Tissues were weighed and then dried at 55°C until a stable weight was recorded. The ratio of wet weight to dry weight was then calculated.

Cytokine ELISA. Cytokines were measured in plasma (1:10 dilution) and lung lavage (1:2 dilution) with sandwich ELISAs using matched pairs (biotinylated and non-biotinylated) of anti-murine antibodies against keratinocyte-derived chemokine (KC or CXCL1) and macrophage inflammatory protein-2{alpha} (MIP-2{alpha} or CXCL2) along with their recombinant proteins (R&D Systems, Minneapolis, MN), as previously described (25). Peroxidase-conjugated streptavidin (Jackson ImmunoResearch Laboratories) and the color reagent TMB were used as the detection system. The reaction was stopped with 1.5 N sulfuric acid, and the absorbance was read at 450 and 630 nm.

Flow cytometry. Samples of blood or lung homogenate were subjected to hypotonic lysis, washed, and resuspended in PBS with 0.1% sodium azide and 1.0% bovine calf serum (HyClone, Logan, UT) at a concentration of 1.0 x 106 cells/ml. Suspensions were incubated for 5 min (4°C) with 0.5 µg Fc{gamma}II/III reagent (BD Pharmingen) to block Fc receptors. The cells were incubated for 30 min at 4°C with 2.5 µg/ml of phycoerythrin-conjugated anti-mouse CXCR2 PE (R&D Systems), fluorescein isothiocyanate-conjugated anti-mouse CD11b (R&D Systems), and allophycocyanin-conjugated anti-mouse Gr-1 (Ly-6G; BD, Pharmingen). Cells were also stained with the appropriate isotype controls (R&D Systems). The fluorescence was measured with a Cytomics FC 500 (Beckman Coulter, Fullerton, CA) flow cytometer using a 488-nm excitation laser with peak emission ranging from 515 nm to 545 nm. Compensation was performed in all experiments utilizing WinList for 32 software (Verity Software House, Topsham, ME).

Immunohistochemistry. Lung sections were deparaffinized and rehydrated. The sections were then incubated with peroxide blocking solution (Super Sensitive Link-Label IHC Detection System; BioGenex, San Ramon, CA) for 10 min. A primary rat anti-mouse CXCR2 (5 µg/ml; R&D Systems) antibody was then allowed to incubate for 1 h followed by incubation with a biotinylated anti-Ig (MultiLink; BioGenex) for 20 min. Negative controls were performed without primary antibody. The sections were then incubated with peroxidase-conjugated streptavidin in PBS followed by a 3,3'-diaminobenzidine solution for 20 min. The slides were counterstained with hematoxylin and mounted for microscopy.

Statistical analysis. Summary data were expressed as the means ± SE. The Student's t-test or ANOVA with post hoc Tukey's test for pairwise comparisons were used for data analysis with GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA). Median lung injury scores from histological evaluations were also compared by the Mann-Whitney test. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Exogenous CO reduced early pulmonary neutrophil recruitment. At 6 h after IT injection, the BAL fluid cell counts of animals given IT saline (n = 10/group) were unaffected by exposure to CO (Fig. 1A). Injection of acid IT (n = 19–20/group) increased total cell counts in the BAL fluid, and this was primarily due to a significant increase in neutrophil counts (Fig. 1B). When exposed to CO immediately after IT injection of acid, neutrophil recruitment to the BAL fluid was significantly reduced (Fig. 1B). These results were supported by the lung injury scores (Fig. 1C). There was no histological evidence that CO had deleterious effects on the lung in the mice that received IT saline. Acid aspiration resulted in a significant increase in the lung injury score by 6 h (Fig. 1C). When mice were exposed to continuous CO for 6 h after aspiration, the injury score was significantly reduced.


Figure 1
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  		Fig. 1. Acute inflammation and injury after acid aspiration. Mice were given intratracheal (IT) injections of saline or acid, with or without continuous exposure to carbon monoxide (CO). Mice were euthanized 6 h after IT injection. Total cell (A) and neutrophil (B) counts were performed on bronchoalveolar lavage (BAL) fluid. C: lung injury scores were based on histological evidence of alveolar congestion, hemorrhage, alveolar wall thickness, and neutrophil accumulation. Cumulative scores for each lung were averaged over groups. CO exposure significantly decreased neutrophil counts and lung injury scores after acid aspiration. N = 10–20/group. #P < 0.05 compared with saline and saline/CO. *P < 0.05 compared with acid.

 
Exogenous CO rapidly affects pulmonary neutrophil recruitment. To further validate the effects of CO, the experiment was repeated with a variation in CO exposure (Fig. 2). After IT acid, animals given CO were exposed for 3 h. After only 3 h of CO exposure, animals (n = 10–11/group) were then placed in room air conditions identical to that of control animals for an additional 3 h. Euthanasia was performed 6 h after the IT injection. Animals exposed to CO for only 3 h also had significantly (P < 0.05) reduced neutrophil numbers in BAL fluid (0.18 ± 0.05 x 106/mouse) compared with those without CO exposure (0.51 ± 0.11 x 106/mouse). This mimicked the effects of continuous exposure for 6 h and suggests that the major effects of CO on neutrophil recruitment occur very early in the exposure.


Figure 2
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  		Fig. 2. BAL fluid cell counts after short-term CO. After IT injections of acid solution, mice were treated for 3 h with low-dose CO and then exposed to room air for an additional 3 h. BAL fluid cell counts showed significantly decreased neutrophil counts, confirming the effects of CO, and demonstrated that beneficial effects occur quickly. N = 10–11/group. *P < 0.05 compared with acid.

 
CO had no cumulative effect on neutrophil recruitment. Although early effects on neutrophil recruitment were documented, aspiration injury can progress beyond 6 h. Therefore, the impact of CO exposure was also examined 24 h after the IT injection. The total cell count in BAL fluid (Fig. 3A) as well as the neutrophil counts (Fig. 3B) showed no significant differences between acid aspiration and acid aspiration treated with CO (n = 8–10/group). Histological examination also showed no differences in lung injury scores between the groups with acid aspiration (Fig. 3C). These results suggest that exogenous CO administered at 500 ppm slowed early neutrophil accumulation in this model of acid aspiration but did not completely inhibit recruitment.


Figure 3
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  		Fig. 3. Progressive inflammation and injury after acid aspiration. Mice were given IT injections of saline or acid, with or without exposure to CO. At 24 h, total cell (A) and neutrophil (B) counts were performed on BAL fluid. C: lung injury scores were based on histological evidence of alveolar congestion, hemorrhage, alveolar wall thickness, and neutrophil accumulation. Although early neutrophil counts were decreased by CO exposure, the neutrophil counts and lung injury at 24 h showed no influence of CO exposure on acid aspiration. N = 10–20/group. #P < 0.05 compared with saline and saline/CO at that time point.

 
CO had no effect on other parameters of lung injury. To further investigate lung injury, albumin levels in the BAL fluid were examined and demonstrated no adverse effects of CO treatment after IT saline (Fig. 4). Acid aspiration caused a dramatic increase in albumin levels. In contrast to the histology scores, the BAL albumin was not affected by continuous CO treatment for 6 or 24 h. Lung wet-to-dry weight ratios also showed no difference when acid aspiration was followed by exposure to 500 ppm of CO for 24 h (Fig. 5).


Figure 4
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  		Fig. 4. Albumin levels in BAL fluid. After IT injections of saline or acid, groups of mice were treated with or without CO exposure. BAL fluid was collected at 6 or 24 h for analysis with an albumin ELISA. For animals with acid aspiration, treatment with CO did not alter the albumin levels in BAL fluid. N = 10–20/group. #P < 0.05 compared with saline and saline/CO at that time point.

 

Figure 5
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  		Fig. 5. Lung wet-to-dry weight ratios. After IT injections of saline or acid, groups of mice were treated with or without CO exposure. Whole lung was collected 24 h later. For animals with acid aspiration, treatment with CO did not alter lung wet-to-dry weight ratio, suggesting no change in vascular integrity. N = 7–8/group. #P < 0.05 compared with saline.

 
CO treatment did not affect local or systemic levels of CXC chemokines. To determine if the early reduction in neutrophil recruitment was related to changes in chemokine levels, the local and systemic levels of two CXC chemokines, KC and MIP-2{alpha}, were evaluated. Within 6 h of IT saline, there were low levels of plasma KC (154.8 ± 17.3 pg/ml) and MIP-2{alpha} (19.9 ± 0.4 pg/ml) that were not significantly altered by CO exposure (121.1 ± 41.2 pg/ml and 35.1 ± 9.1 pg/ml, respectively). Acid aspiration increased the plasma levels of KC (1,585.3 ± 429.2 pg/ml) but did not substantially alter levels of MIP-2{alpha} (93.0 ± 54.6 pg/ml). The plasma KC levels remained elevated (1,398.8 ± 320.0 pg/ml) with CO treatment. Likewise, KC and MIP-2{alpha} levels in BAL fluid were negligible after saline, and exposure to CO did not affect these levels (Fig. 6, A and B). The BAL fluid levels of both KC and MIP-2{alpha} were increased 6 h after acid aspiration (Fig. 6, A and B), with KC concentrations consistently higher than those of MIP-2{alpha}. CO treatment after aspiration did not reduce the concentrations of CXC chemokines. In fact, the BAL fluid concentrations in animals treated with CO after aspiration of acid were higher than those without CO. The chemokine levels in BAL fluid subsided by 24 h, except for MIP-2{alpha} levels retained by the aspiration group that was not treated with CO.


Figure 6
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  		Fig. 6. CXC chemokines in BAL fluid. Mice were given IT injections of saline or acid with or without immediate treatment of spontaneously inhaled CO (500 ppm). BAL fluid samples collected at 6 and 24 h after aspiration were analyzed for the CXC chemokines KC (A) and MIP-2{alpha} (B). Acid aspiration increased the chemokine levels, but treatment with CO did not cause a reduction of those levels. N = 10–20/group. #P < 0.05 compared with saline and saline/CO for that time point.

 
Exogenous CO did not alter CXCR2 protein expression. The expression of receptors for the CXC chemokines will affect neutrophil recruitment. Therefore, the protein expression of CXCR2 in lung tissue and on peripheral neutrophils was investigated. At 6 h after IT saline, immunohistochemistry revealed low-level expression of CXCR2 on the epithelium of the bronchioles (Fig. 7B) as well as endothelium and alveoli. Acid aspiration caused a relative increase in CXCR2 expression in all of these cell types. Negative controls performed on tissues from the same animals confirmed no increase in nonspecific background staining after aspiration (Fig. 7B). Of particular note, there was strong expression of CXCR2 on the endothelium in blood vessels (Fig. 7C). This pattern of expression did not change with CO exposure. CXCR2 protein expression was still strong in all cell types and still very evident on endothelium (Fig. 7).


Figure 7
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  		Fig. 7. CXCR2 protein expression in lung tissue. Lung tissue was harvested from mice 6 h after IT injections of saline or acid with or without subsequent exposure to CO. Tissues were processed for immunohistochemistry. Compared with a negative control (A), slight expression of CXCR2 expression (B; brown color indicated by arrowheads) was evident on the endothelium of animals given IT saline. With acid aspiration, there was marked expression of CXCR2 on endothelium (C, arrowheads) and epithelial cells. The pattern of expression was not altered by CO exposure (D). Photomicrographs (x1,000) are representative of results from 4 mice/group. Scale bar, 50 µm.

 
Peripheral blood neutrophils were also evaluated by flow cytometry 6 h after aspiration. Neutrophils were identified by characteristic forward and side scatter patterns as well as Gr-1 staining. The mean fluorescence intensity of peripheral blood neutrophils with surface expression of CXCR2 was not changed when acid aspiration was followed by continuous CO exposure (data not shown). Likewise, the percentage of peripheral blood neutrophils expressing surface CXCR2 within 6 h of aspiration (78.1 ± 3.2% of total peripheral neutrophils) was unchanged by exogenous CO (76.8 ± 3.9% of total peripheral neutrophils).

Exogenous CO affects peripheral white blood cell counts. Previously, we have shown that peripheral neutrophil counts will increase after acid aspiration (23). To determine if CO treatment limited this response, complete blood counts were obtained before euthanasia at 6 or 24 h after aspiration. The total and differential white blood cell (WBC) counts from animals given IT saline appeared unaffected by CO treatment (Fig. 8, A–D). Animals given IT injections of acid demonstrated a significant increase in peripheral blood neutrophils within 6 h of the lung insult compared with those given saline (Fig. 8C). These counts had decreased by 24 h after acid aspiration but still remained significantly increased compared with animals given IT saline. In animals given IT acid, exposure to CO was associated with an additional increase in the peripheral neutrophil counts within 6 h (Fig. 8C). Therefore, the reduction in BAL neutrophil recruitment could not be explained by inhibition of the systemic response to aspiration. Increases in monocyte and lymphocyte counts were also evident when the animals with acid lung injury were exposed to CO, contributing to an increase in total WBC counts (Fig. 8, A, B, and D). However, the effect of CO exposure on peripheral blood counts was not evident at 24 h after acid insult.


Figure 8
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  		Fig. 8. Cell counts in peripheral blood. Aspiration of either saline or acid was treated with or without exposure to CO. Peripheral blood counts of total white blood cells (WBC) (A), monocytes (B), neutrophils (C), and lymphocytes (D) were obtained by an automated analyzer at 6 and 24 h after IT injections. Aspiration increased monocyte and neutrophil counts by 6 h. Treatment with CO further increased all the cell counts. This effect was not evident at 24 h. N = 10–20/group. #P < 0.05 compared with saline and saline/CO at the same time point. *P < 0.05 compared with acid at the same time point.

 
Exogenous CO affects neutrophil activation. Flow cytometry was used to evaluate the presence of CD11b on the surface of peripheral blood neutrophils within 6 h of aspiration. Of the total cell population counted, the percentage of CD11b-positive neutrophils in the peripheral blood was significantly increased after acid aspiration compared with normal, control animals. CO exposure significantly decreased the percentage of CD11b-positive neutrophils detected after acid aspiration (Fig. 9). Similarly, a significant decrease (P < 0.05) in the percentage of peripheral neutrophils expressing CD11b was found when animals (n = 10/group) with acid aspiration (28.0 ± 3.4%) were exposed to 3 h of CO followed by 3 h of room air (18.0 ± 2.7%).


Figure 9
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  		Fig. 9. CD11b expression on neutrophils in peripheral blood. Mice with acid aspiration were treated with or without exposure to CO. Peripheral blood was obtained for flow cytometry at 6 h. CD11b surface expression was examined by gating on neutrophils (Gr-1+ cells). Compared with peripheral blood taken from normal, control animals, the percentage of CD11b-positive neutrophils was significantly increased by acid aspiration. However, the percentage of neutrophils expressing CD11b was significantly decreased when animals were exposed to CO. N = 6 aspirations/group and 3 control animals. *P < 0.05 compared with acid.

 
The activation of neutrophils, assessed by CD11b surface expression, was determined by flow cytometry of lung digests taken 6 h after aspiration. The CD11b-positive neutrophils represented a significantly higher percentage of the cells counted from the lung digests after aspiration of acid compared with saline (Fig. 10). Although exposure to CO did decrease the percentage of CD11b-positive cells in the lung after acid aspiration, this difference was not statistically significant (Fig. 10).


Figure 10
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  		Fig. 10. CD11b expression on neutrophils in lung tissue. Mice with saline or acid aspiration were treated with or without exposure to CO. After BAL and perfusion, lung tissue was digested with collagenase and analyzed via flow cytometry. The percentage of cells in lung digests that had surface expression of CD11b and Gr-1 was significantly increased after acid aspiration compared with saline. With CO exposure, the percentage of CD11b-positive neutrophils was decreased, although the change was not statistically significant. N = 6/group. #P < 0.05 compared with saline and saline/CO groups.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, continuous administration of inhaled exogenous CO decreased the recruitment of neutrophils to the lung during the first 6 h after acid aspiration. These results demonstrate that CO can modulate the early, acute response to a direct lung insult even without pretreatment. Previous reports documented reduced neutrophil recruitment in response to CO in models of lung injury that developed over several hours to days after the initiation of the insult. Otterbein et al. (29) demonstrated a marked decrease in the number of neutrophils found in the airways when mice were exposed to 84 h of hyperoxia and 250 ppm of CO. In a lung transplant model, Song et al. (36) showed that exposure to 500 ppm CO decreased lung MPO activity in rats 6 days after orthotopic lung transplantation. Exogenous CO also decreased the numbers of inflammatory cells found in lungs 24 h after the final challenge in an ovalbumin model of allergic lung injury (4). Interestingly, experimental studies have rarely addressed the effects of CO on the relatively acute development of injury in response to a direct lung insult. Similar to our results, one study demonstrated that CO obtunded neutrophil recruitment after 4 h of mechanical ventilation; however, the direct injury was first primed with intravenous LPS, and progression of the injury beyond 4 h was not examined (7). In a study with conflicting results, pretreatment followed by continuous treatment with CO for 4 h did not affect lung inflammation or pulmonary neutrophil activation after direct exposure to intratracheal LPS (10). As suggested in that study, the lack of effectiveness may have been due to the severity and/or hyperacute response of the LPS model. The acid aspiration used in our study induces a nonlethal lung injury with neutrophil recruitment that increases between 3 and 8 h after administration. With this model, neutrophil counts in BAL tend to remain high for 24 h after untreated aspiration (23). The slower onset of injury may explain why CO demonstrated some efficacy in reducing early neutrophil recruitment in this model compared with the previously reported LPS model.

Neutrophil trafficking is dependent on a number of factors affecting activation, chemoattraction, and attachment of the cells. The specific mechanisms by which CO can affect neutrophil migration from the peripheral blood to the lung tissue and airways have not been fully characterized. Studies have shown that the anti-inflammatory effects of CO stem largely from the enhanced activation of MAP kinases in macrophages (27) and in injured lung tissue (7). Macrophages display a rapid decrease in TNF-{alpha} and IL-6 production with a concurrent increase in IL-10 production (27), factors that could affect neutrophil activation. In addition, CO may regulate the sequestration of neutrophils through molecules such as the intercellular adhesion molecule-1 (ICAM-1). The expression of ICAM-1 on endothelial cells allows firm attachment to β-integrins on the surface of rolling neutrophils, promoting their migration from the microvasculature into tissues (37). Previous reports have documented that the expression of adhesion molecules including ICAM-1 (35) is modulated by HO-1 and that CO-releasing molecules attenuate burn-associated lung injury by reducing ICAM-1 expression (38).

The results of our studies suggested another adhesion molecule, CD11b, could be affected by administration of inhaled CO. CD11b, in combination with CD18, is a β-integrin stored in the cytoplasmic granules of WBC. Upon stimulation, cells will rapidly upregulate CD11b by mobilizing cytoplasmic stores; therefore, CD11b is a marker of cellular activation and mediates neutrophil adherence to ICAM-1 on endothelial cells (37). In our study, CO was associated with lower percentages of neutrophils expressing CD11b, particularly in peripheral blood. One explanation for this could be that the reduction in CD11b-expressing neutrophils in peripheral blood was due to migration of the neutrophils from the circulating pool to the site of inflammation during CO treatment. To rule out this possibility, we examined whole lung homogenates for neutrophils expressing CD11b and found no evidence that excessive numbers were sequestered in the CO-treated animals compared with the untreated animals. In fact, the percentage of neutrophils expressing CD11b in the lung was decreased, although this was not a significant difference. Consequently, it would appear that inhaled CO treatment has an effect on the activation of peripheral blood neutrophils. This would offer another mechanism for the decrease in pulmonary inflammation seen with CO exposure. It would also help explain the increase in absolute numbers of neutrophils found in the peripheral blood after exposure to CO. It is doubtful that the neutrophilia seen at 6 h represented CO toxicity since the cell counts did not worsen but returned to normal with additional exposure up to 24 h. Other studies have shown that CO actually decreased granulocyte/macrophage colony-stimulating factor production by macrophages (32) and had no effect on bone marrow content of other inflammatory cells in an allergy model (4). This suggests that the neutrophilia seen in our study was not secondary to release from the bone marrow. We speculate that the increase in peripheral blood neutrophils as well as the decrease in BAL fluid neutrophils associated with CO treatment was due to a decrease in the expression of adhesion molecules. Our results suggest that surface expression of adhesion molecules on peripheral neutrophils decreases in addition to the previously reported decrease in adhesion molecules noted on endothelial cells.

Although CO may regulate the expression of CD11b and/or ICAM-1, the benefits of this mechanism may be somewhat restricted in acid aspiration lung injury (11, 19). Studies have demonstrated that acid aspiration is mediated by both CD11b/CD18-dependent and -independent mechanisms. In a rabbit model of aspiration, CD18 and ICAM-1 were not involved at the site of direct acid injury but did mediate recruitment in other areas of the lung (11). A similar study in rats given anti-CD11b antibody demonstrated no affect on recruitment in focal areas of direct acid injury, whereas recruitment was reduced in the contralateral lung (19). Therefore, any beneficial effects of CO on CD11b or ICAM-1 expression would be restricted to specific areas of the lung. This might explain why the effects of CO were significant at 6 h but less evident after 24 h in our model of acid aspiration lung injury. It is possible that neutrophil recruitment was reduced in parts of the lung but unaffected in other parts, effectively slowing but not halting neutrophil recruitment. This effect would be particularly evident if chemotactic signaling was unaffected by CO treatment.

Surprisingly, it appears that production of a major type of neutrophil chemoattractant involved in acid aspiration (8, 23, 33), the CXC chemokine, is not influenced by CO. A previous in vitro study showed no influence of exogenous CO on LPS-induced MIP-2{alpha} or KC production in a macrophage cell line (7). Likewise, there was no change in MIP-2{alpha} levels in the BAL fluid of mice after IT administration of LPS (10); however, there was also no change in neutrophil recruitment in that study. Our study further substantiates that, although CO may influence cell recruitment, reduced production of CXC chemokines is not the mechanism for the decrease in neutrophil recruitment in vivo. In our study, CXC chemokine concentrations in BAL fluid were actually slightly higher when aspiration was treated with CO compared with untreated aspiration. These higher concentrations of chemokines in the CO-treated animals could be the result of lower neutrophil counts that would effectively reduce the availability of CXCR2 binding sites. Despite higher BAL fluid concentrations, CO could still influence the recruitment of neutrophils if the chemokine gradients favored retention of the cells in the peripheral blood and not the lung (3). When the ratios of BAL fluid to plasma chemokines that occurred 6 h after aspiration were compared, there was no influence of CO treatment on the chemokine ratios (data not shown). Therefore, the CXC chemokines could provide an ongoing stimulus for recruitment of neutrophils via a CD11b/ICAM-1-independent mechanism in the areas of the lung directly exposed to the acid insult.

Although early lung inflammation was significantly reduced by CO in our study, exogenous CO did not affect the acute accumulation of albumin in the airways, an indicator of compromised vascular integrity. Recent evidence suggests that the chemokine receptor CXCR2 has a significant role in the development of vascular permeability associated with acute lung injury (2, 30). Specifically, the presence of CXCR2 on endothelial cells was of greater significance than CXCR2 on neutrophils with regard to developing protein leakage in an LPS model of acute lung injury (30). Stimulation of the receptor on in vitro pulmonary endothelial cells produced dose-dependent cytoskeletal remodeling that may explain the link between CXCR2 and vascular integrity (30). In our studies, the increased expression of CXCR2 protein in lung tissues was evident on endothelial, epithelial, and alveolar cells after acid aspiration. This pattern was still evident after exposure to exogenous CO. Since chemokine levels were not affected by the treatment, this potential mechanism for vascular leakage remained intact. Our finding of sustained vascular permeability despite a reduction in neutrophil recruitment is similar to that seen after CO treatment of combined ventilation and LPS injury (7). Likewise, a study of lung inflammation induced after cutaneous burn injury demonstrated that the IV administration of CO releasing molecules reduced lung inflammation but did not affect BAL fluid albumin or lung wet-to-dry weight ratios (38). Although studies of hyperoxic lung injury did show changes in lung protein (28, 29), our study and others (7, 38) suggest that the anti-inflammatory effects of CO do not affect vascular integrity in acute injury.

Although early recruitment was decreased, neutrophil accumulation in the lung and airways appeared to be progressive after acid aspiration regardless of exposure to CO. However, it is important to note that our studies examined a limited range of CO dosing in a fixed model of lung injury. The amount of carboxyhemoglobin in blood is dependent on the concentration of CO delivered and the exposure time (12). Studies have shown that concentrations up to 1,000 ppm are tolerated in humans (12) and mice (14). Concentrations of 3,000 ppm were lethal in a mouse model of CO-induced myocardial dysfunction (14), whereas another study suggested that a concentration of 10,000 ppm caused lethality (28). The majority of pulmonary studies has demonstrated efficacy with 250 (4, 7, 22, 28, 29) or 500 (5, 10, 36) ppm of CO delivered by either mechanical ventilation or spontaneous inhalation (12). Many of these studies use protocols that involve pretreatment with CO (4, 10, 22, 29). Our study used the highest of the commonly reported doses without a pretreatment because this would be more relevant to the majority of clinical aspiration cases. However, higher dosages of CO could potentially be used safely and might be even more efficacious than 500 ppm in some situations. We did examine two different CO exposure times (3 or 6 h) and demonstrated similar decreases in neutrophil recruitment at 6 h postaspiration with both treatments. It is possible that intermittent administration of CO over short exposure times would be beneficial, and this strategy has proven effective for reducing airway hyperresponsiveness in an asthma model (1). It is also likely that the degree of injury would dictate the relative efficacy of CO treatment. Since the amount of lung injury is dependent on the volume and pH of the aspirate (16), we optimized these factors to give a mild, nonlethal but reliable model of lung inflammation. However, inflammatory responses may also vary with the age, gender, and genetic background of the mouse (6), suggesting that a number of additional studies would be required to completely optimize the effectiveness of CO treatment after aspiration. The present studies cannot rule out the possibility that CO would be even more effective in models producing less inflammation or more focal injury. In any case, it would appear important to examine the effects of CO treatment at early and later time points to fully evaluate acute lung injury models.

In conclusion, CO did reduce early neutrophil recruitment to the airways and histological signs of lung injury in this model of acid aspiration. Pretreatment was not required to significantly obtund neutrophil recruitment. The effects of CO on neutrophil recruitment were accompanied by a significant decrease in the percentage of peripheral blood neutrophils and slight reduction in lung-sequestered neutrophils with surface expression of the activation marker CD11b. In spite of CO treatment, the lung inflammation was progressive over 24 h. This may be due to the fact that acid aspiration lung injury can be induced by CD11b-independent mechanisms and that chemotactic signaling through the CXC chemokines remains intact after CO treatment. Despite this, it appears that CO has some efficacy in this type of acute lung injury by slowing the accumulation of neutrophils in the airways. Further studies are needed to find the most advantageous regimen of CO and define the indications for its use after acid aspiration.


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This work was supported by National Institute of General Medical Sciences Grants GM-65486 and GM-067189.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Nemzek, Univ. of Michigan, Unit for Laboratory Animal Medicine and Dept. of Pathology, 018 ARF, 1150 W. Medical Center Drive, Ann Arbor, MI 48109 (e-mail: jnemzek{at}umich.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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D. Morse and A. M. K. Choi
Inhaled CO in the treatment of acute lung injury
Am J Physiol Lung Cell Mol Physiol, April 1, 2008; 294(4): L642 - L643.
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