AJP - Lung Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 289: L807-L815, 2005. First published June 10, 2005; doi:10.1152/ajplung.00477.2004
1040-0605/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/5/L807    most recent
00477.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (20)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Reutershan, J.
Right arrow Articles by Ley, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Reutershan, J.
Right arrow Articles by Ley, K.

Sequential recruitment of neutrophils into lung and bronchoalveolar lavage fluid in LPS-induced acute lung injury

Jörg Reutershan,1,4 Abdul Basit,2 Elena V. Galkina,1,3 and Klaus Ley1,2,3

1Robert M. Berne Cardiovascular Research Center, Departments of 2Physiology and Biological Physics, and 3Biomedical Engineering, University of Virginia, Charlottesville, Virginia; and 4Department of Anesthesiology and Intensive Care Medicine, University of Tübingen, Tübingen, Germany

Submitted 23 December 2004 ; accepted in final form 9 June 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Infiltration of activated neutrophils [polymorphonuclear leukocytes (PMN)] into the lung is an important component of the inflammatory response in acute lung injury. The signals required to direct PMN into the different compartments of the lung have not been fully elucidated. In a murine model of LPS-induced lung injury, we investigated the sequential recruitment of PMN into the pulmonary vasculature, lung interstitium, and alveolar space. Mice were exposed to aerosolized LPS and bronchoalveolar lavage fluid (BAL), and lungs were harvested at different time points. We developed a flow cytometry-based technique to assess in vivo trafficking of PMN in the intravascular and extravascular lung compartments. Aerosolized LPS induced consistent PMN migration into all lung compartments. We found that sequestration in the pulmonary vasculature occurred within the first hour. Transendothelial migration into the interstitial space started 1 h after LPS exposure and increased continuously until a plateau was reached between 12 and 24 h. Transepithelial migration into the alveolar air space was delayed, as the first PMN did not appear until 2 h after LPS, reaching a peak at 24 h. Transendothelial migration and transepithelial migration were inhibited by pertussis toxin, indicating involvement of G{alpha}i-coupled receptors. These findings confirm LPS-induced migration of PMN into the lung. For the first time, distinct transmigration steps into the different lung compartments are characterized in vivo.

polymorphonuclear leukocytes; pulmonary circulation; chemokines; lipopolysaccharide; flow cytometry; pertussis toxin

ACUTE LUNG INJURY (ALI) and acute respiratory distress syndrome (ARDS) are characterized by a disturbance of the alveolar-capillary barrier associated with several clinical disorders. There is no specific therapy, and the mortality of this disease is still high. Our current understanding of the molecular mechanisms of ALI/ARDS has recently been described as "embryonic at best" (27).

Migration of activated polymorphonuclear leukocytes (PMN) plays a key role in development of ALI and ARDS (1). Here, we investigate the sequential migration steps from blood to air space (intravascular sequestration, transendothelial migration, transepithelial migration).

A variety of stimuli induce PMN migration into the lung. Endotoxin of gram-negative bacteria [lipopolysaccharide (LPS)] induces a range of inflammatory responses. Toll-like receptor-4 (TLR4) is the most important cellular receptor for LPS. LPS stimulates the response to chemoattractants and increases PMN migration at sites of inflammation (14). TLR4 is essential for LPS-induced PMN migration into the lung as shown by the absence of a response in TLR4-deficient mice (3). In the lung, the response to LPS is regulated by radioresistant cells, most likely endothelial cells (2) or alveolar macrophages (28).

The administration of LPS alone might not reflect the whole complexity of the human disease, because it does not consider preexisting diseases, fluid resuscitation, or mechanical ventilation (36). However, infections with gram-negative bacteria and exposure to their predominant pathogenic component play a key role in both development and outcome of ARDS (26).

PMN trafficking into the vascular compartment of the lung, also known as "margination" (9), and into the bronchoalveolar space has been studied extensively in various models of ALI. Adhesion molecules and CXC chemokines have been shown to be involved. CXC chemokines, such as CXCL8 (IL-8), promote PMN migration into the alveolar compartment (29), and pertussis toxin (PTx)-dependent chemokine receptors are essential for PMN infiltration in the lung (4, 42). Selectins and integrins are thought to be required for PMN sequestration into the vascular compartment (6). However, results from studies using monoclonal antibodies and mutant mice have yielded conflicting results (10). The importance of investigating each step of PMN migration in the lung has been emphasized recently (8).

Methodological limitations complicate the assessment of PMN trafficking in the lung. Most studies employ indirect parameters to assess PMN trafficking in the lung. For instance, the drop in circulating PMN counts in response to an inflammatory stimulus was used to estimate PMN sequestration in the lung vasculature (6, 31). However, recruitment to other organs might occur at the same time. Ex vivo labeling of murine PMN might result in neutrophil activation that makes results uninterpretable (7). Myeloperoxidase activity in the lung is often measured to estimate PMN infiltration, but this technique is not able to distinguish between intravascular and interstitial PMN. Intravital microscopy of the pulmonary microcirculation has recently become available in mice (35, 39) and will promote insights into the interactions between leukocytes and endothelium. However, this technique is technically challenging because of the respiratory movement, requires mechanical ventilation, and allows observation of only the most superficial lung capillaries, which may not be representative of the whole lung. Morphometric analysis, such as electron microscopy (16), is useful but remains semiquantitative, time consuming, and expensive.

In this study, we developed a flow cytometry-based approach to assess the different steps of PMN trafficking in a murine model of LPS-induced ALI. PMN accumulation in the pulmonary vasculature, transendothelial migration into the interstitium, and transepithelial migration from the interstitium into the air space occurred as a sequential process in a time-dependent manner. Our findings improve the current understanding of neutrophil recruitment into the inflamed lung and airways in a model that mimics some aspects of ALI/ARDS.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Mice. Wild-type male C57BL/6 mice were obtained from Jackson Labs (Bar Harbor, ME). All animal experiments were approved by the Animal Care and Use Committee of the University of Virginia. Mice were 8–12 wk of age.

Antibodies for flow cytometry. Rat anti-mouse antibodies for flow cytometry were obtained from Pharmingen (anti-CD45, clone 30-F11), Caltag (anti-7/4, recognizing the 7/4 antigen on murine neutrophils), and eBioscience (anti-TER-119, recognizing glycophorin A-associated TER-119 on cells of the erythroid lineage). Anti-mouse GR-1 antibody was purified from supernatant of the GR-1 hybridoma (ATCC) by the biomolecular facility of University of Virginia. GR-1 was labeled with a staining kit following the manufacturer's directions (Alexa Fluor 633, Molecular Probes). Appropriate rat anti-mouse IgG2a and IgG2b (Pharmingen) were used as isotype controls.

Murine model of ALI. Aerosolized LPS was utilized to induce PMN infiltration in the lung (40). Besides PMN migration, LPS inhalation is known to induce the expression of various inflammatory mediators such as chemokines and adhesion molecules. LPS has also been shown to increase airway resistance (24). Up to four mice were exposed simultaneously to aerosolized LPS in a custom-built cylindrical chamber (20 cm in length, 9 cm in diameter) connected to an air nebulizer (MicroAir; Omron Healthcare, Vernon Hills, IL). This system produced particles in the range of 1–5 µm. LPS from Salmonella enteritidis (Sigma, St. Louis, MO) was dissolved in 0.9% saline (500 µg/ml), and mice were allowed to inhale LPS for 30 min. One side of the chamber was connected to a vacuum pump, and a constant flow rate of 15 ml/min was ensured by a flow meter (Gilmont Instruments, Barrington, IL).

PMN counts in bronchoalveolar lavage fluid and lung tissue. At different time after LPS exposure, mice were anesthetized with an intraperitoneal injection of ketamine (125 mg/kg; Sanofi Winthrop Pharmaceuticals, New York, NY), xylazine (12.5 mg/kg; Phoenix Scientific, St. Joseph, MO), and atropine sulfate (0.025 mg/kg; Fujisawa, Deerfield, IL). The pulmonary circulation was rinsed by injection of 10 ml of PBS at 25 cmH2O into the beating right ventricle after the inferior vena cava had been cut to allow exsanguination. This was done to remove nonadherent PMN from the pulmonary vessels. The trachea was cannulated (22 GA Insyte, Becton Dickinson), and 1 ml of PBS was infused intratracheally and withdrawn. This procedure was repeated six times, resulting in a total volume of 7 ml. Bronchoalveolar lavage (BAL) fluid was centrifuged for 5 min at 300 g. The pellet was resuspended in 1 ml of buffer (1% BSA and 0.1% sodium azide in PBS), and a 10-µl aliquot was used for cell count with a hemocytometer (Trypan blue exclusion).

After performing BAL, we harvested lungs en bloc. Mediastinal tissue was removed, and lungs were minced and digested with 125 U/ml collagenase type XI, 60 U/ml hyaluronidase type I-s, and 60 U/ml DNase (all Sigma) at 37°C for 30 min. Digested lungs were passed through a 70-µm cell strainer (BD Falcon, Bedford, MA), and the resulting cell suspension was centrifuged for 10 min at 300 g. The pellet was lysed with 0.83% NH4Cl to remove erythrocytes and centrifuged again. Pellet was resuspended in buffer, and cells were counted with a hemocytometer.

PMN were identified by 1) their typical appearance in the forward scatter-side scatter (FSC/SSC), 2) their expression of CD45+, and 3) two independent PMN markers, 7/4 and GR-1 (19), and the absolute numbers of leukocytes (CD45+) and PMN were calculated. Appropriate isotype antibodies were used to set the gates. All studies were performed on a FACS Calibur (Becton Dickinson, San Jose, CA), and data were analyzed with FlowJo software (Tree Star, Ashland, OR). To confirm the presence of PMN within the different populations as defined by flow cytometry, we sorted both 7/4+GR-1+ and 7/4+GR-1– cells (FACS Vantage, Becton Dickinson) and characterized them morphologically by cytospin (Diff Quick staining, IMEB).

In vivo trafficking experiments. Dialyzed Alexa 633-labeled rat anti-mouse GR-1 (10 µg) antibody was injected intravenously and allowed to circulate for 5 min to bind to intravascular PMN. After 5 min, mice were killed. BAL was obtained as described above, and the lungs were homogenized in the presence of excess unlabeled anti-GR-1 to prevent possible binding of excess Alexa 633-GR-1 to extravascular PMN. Cell suspensions from BAL and lung tissue were made, and cells were counted in a hemocytometer.

Nonperfused or occluded vessels in the lung might result in trapped neutrophils, not accessible for the injected antibody. This would lead to an underestimation of PMN counts in the intravascular compartment. Nonperfused/occluded vessels contain white and red blood cells. To assess the significance of this phenomenon in our model, a monoclonal antibody to the TER-119 antigen on erythrocytes was injected intravenously (TER-119; eBioscience, San Diego, CA). This 52-kDa molecule is associated with glycophorin A on cells of the erythroid lineage (22). Five minutes after injection, blood and lungs were harvested. Erythrocytes were defined by their typical appearance in the forward-sideward scatter and the amount of TER-119+ erythrocytes in each organ was expressed as percentage of total erythrocytes by flow cytometry. To assess the effect of intravenous injection of anti-GR-1 on peripheral PMN counts, in some experiments, blood was withdrawn from the tail vein and blood counts were performed before and 10 min after antibody injection with an automatic cell counter (Hemavet; Drew Scientific, Dallas, TX). In all experiments, animals exposed to aerosolized saline served as control.

Cytospin of BAL. In some experiments, cytospins of the BAL (without LPS treatment and 24 h after LPS inhalation) were performed with a cytocentrifuge (Shandon, Southern Sewickley, PA). The cytospun cells were Giemsa stained, air-dried, and coverslipped.

Lung permeability. To confirm lung injury in our LPS inhalation model, we determined microvascular permeability using the Evans blue dye extravasation technique. Evans blue (20 mg/kg iv, Sigma-Aldrich) was injected 30 min before death. Lungs were perfused through the spontaneously beating right ventricle. Lungs were removed, and Evans blue was extracted as described previously (32). The absorption of Evans blue was measured at 620 nm (A620) and corrected for the presence of heme pigments: A620 (corrected) = A620 – (1.426 x A740 + 0.030) (45). Extravasated Evans blue was determined 6 h after LPS or saline inhalation and calculated against a standard curve (micrograms Evans blue dye per gram lung).

PTx pretreatment. Chemokines have been shown to regulate PMN migration in the lung (42). To block chemokine-mediated PMN migration, some mice received tail vein injections of 4 µg of PTx from Bordetella pertussis (lyophilized powder, Sigma) 30 min before LPS exposure. This dose completely inhibits G{alpha}1-mediated signaling (41). PTx was dissolved in physiological saline. PMN in lung and BAL were assessed 14 h after LPS exposure. In addition, a dose-response curve was established for each lung compartment 12 h after LPS exposure (0, 0.04, 0.4, or 4 µg PTx/mouse).

Statistical analysis. Data were analyzed with the Excel software package (Microsoft). PMN counts were compared by paired Student's t-test. P < 0.05 was considered statistically significant. Data were expressed as means ± SE.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
LPS-induced PMN recruitment into lung and BAL. LPS inhalation induced a time-dependent PMN recruitment into lung and BAL (Fig. 1). In the lung, significant numbers of leukocytes (CD45+ cells) were present even before LPS administration, and their numbers increased only moderately from four million to a maximum of nine million cells at 4 h after LPS. Neutrophils were also present in resting lungs (approximately one million per mouse), consistent with the concept of a physiologically marginated pool in the pulmonary vasculature. Lung neutrophil numbers reached more than six million cells/mouse at 4 h of LPS administration, which is severalfold more than the total number of all circulating neutrophils consistent with a previously described release of PMN from the bone marrow (37, 46). At the peak of the response, neutrophils accounted for 74 ± 7% of all leukocytes in the lung.



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 1. LPS inhalation induces a time-dependent recruitment of leukocytes and polymorphonuclear leukocytes (PMN) into the lung (A) and bronchoalveolar lavage (BAL, B). X-axis: time after LPS exposure. Absolute numbers of leukocytes (CD45+) and PMN (CD45+GR-1+7/4+) were obtained as the product of flow cytometry percentage and hemocytometer-based total cell counts. Data are presented as means ± SE of n = 4 experiments at each time point.

 
No PMN were observed in the BAL at 0 h. PMN recruitment into the air space was delayed, and the first PMN did not appear until 2 h. Between 2 and 4 h, neutrophil recruitment was very pronounced. After 48 h, cell counts in BAL were reduced but did not reach baseline.

In vivo GR-1 labeling. In the lung homogenate, GR-1 labeling was utilized to distinguish between PMN derived from the pulmonary vasculature (GR-1+7/4+) and PMN derived from interstitial space (GR-1–7/4+). We assessed the PMN labeling 5 min after injection of anti-GR-1 antibody. We found that almost all blood PMN (99.2 ± 0.4%) had been stained with GR-1 5 min after antibody injection (Fig. 2, A and B; Table 1). To test whether GR-1 antibody was leaking into the BAL, GR-1 labeling of PMN in BAL (CD45+7/4+) was assessed at different time points after LPS exposure, 5 min after antibody injection. No GR-1+ cells were found in BAL. When GR-1 antibody was added after BAL harvesting, all PMN were GR-1+ (positive control) (Fig. 2, C and D; Table 1). GR-1+ PMN did not appear in the BAL until 4 h after antibody injection (data not shown).



View larger version (36K):
[in this window]
[in a new window]
  		Fig. 2. In vivo anti-GR-1 labeling. Rat anti-mouse GR-1 (10 µg) was injected, and blood was taken 5 min later. PMN were identified by their typical appearance in the forward-side scatter (A) and by the expression of 7/4 antigen. GR-1 labeling in these cells was determined (B). Five minutes after antibody injection, almost all PMN (>99%) were GR-1+. Plot shows a representative result of n = 6 experiments. SSC, side scatter; FSC, forward scatter. C and D: GR-1 labeling in BAL. At different time points after LPS exposure, 10 µg of rat anti-mouse GR-1 were injected, and BAL was performed 5 min thereafter. PMN were identified by expression of CD45 and 7/4. No GR-1+ cells were found in the BAL (C). D: positive control (BAL stained with anti-GR-1 and anti-7/4 after harvesting). Graphs show representative dot plot 24 h after LPS exposure.

 

View this table:
[in this window]
[in a new window]
  		Table 1. In vivo neutrophil labeling

 
Although all circulating PMN were shown to be GR-1+, potential non- or poorly perfused areas of the pulmonary vasculature might be inaccessible for an intravenously injected antibody. This would result in an underestimation of the intravascular or an overestimation of the interstitial PMN concentration in the lung, respectively. This effect might occur particularly in the injured lung. We therefore labeled erythrocytes by intravenous injection of an antibody to the TER-119 antigen of red blood cells intravenously 24 h after LPS exposure. Red blood cells are not found in the lung interstitium or BAL (5). The amount of TER-119+ cells among all erythrocytes was then determined in both blood and lung homogenate. Five minutes after injection, we found 98.8% of all blood erythrocytes to be TER-119+. At the same time, 97.7% of all erythrocytes were TER-119+ in the lung homogenate, indicating that the injected antibody is able to bind to almost all red cells in the pulmonary vasculature, suggesting that nearly all vessels were perfused.

Effect of anti-GR-1 antibody on PMN blood counts. GR-1 antibody can induce severe neutropenia when given at high doses (33). Therefore, we performed peripheral blood counts before and 10 min after antibody injection. GR-1 injection did not affect blood PMN counts at the concentration used in our study (0.89 ± 0.14 x 103/µl before, 0.86 ± 0.16 x 103/µl after injection; P = 0.90).

In vivo trafficking experiments. We assessed the concentration of PMN in the different lung compartments at 0, 1, 2, 4, 12, and 24 h after LPS exposure. At each time point, anti-GR-1 antibody was injected 5 min before death. PMN were identified by flow cytometry (FSC/SSC gate, CD45+7/4+), and GR-1 was utilized to distinguish between intravascular (GR-1+) and interstitial (GR-1–) PMN (Fig. 3). Both populations predominantly consisted of PMN as confirmed morphologically by cytospins (7/4+/GR-1+, 99% PMN; 7/4+GR-1–, 97% PMN; data not shown). At 0 h, 86% of all PMN were found in the vascular compartment. LPS induced PMN accumulation in the pulmonary vasculature. Both absolute and relative PMN counts increased rapidly until a peak was reached after 4 h. After 4 h, PMN counts in the vasculature decreased and returned to baseline at 24 h after LPS exposure (Fig. 3A).



View larger version (30K):
[in this window]
[in a new window]
  		Fig. 3. In vivo PMN trafficking. A: PMN numbers in the different lung compartments 0, 1, 2, 4, 12, and 24 h after LPS exposure. GR-1 antibody was injected 5 min before death. PMN were identified as 7/4+. GR-1 labeling was used to distinguish between intravascular (GR-1+, top right quadrants) and interstitial (GR-1–, bottom right quadrants) PMN. PMN accumulation in the pulmonary vasculature started rapidly after LPS exposure and reached a peak after 4 h. Significant migration into the interstitial space started between 2 and 4 h after LPS exposure, increased continuously, and reached a plateau after 12 h. Interstitial PMN (%) is indicated in bottom right square. B: PMN in BAL. Initial PMN concentration at time (t) = 0 is negligible. A significant increase in PMN concentration appears between 2 and 4 h. PMN in the BAL do not stain for GR-1 as this antibody has been injected intravenously and does not reach the alveolar air space. Graphs are representative of n = 4 time-course experiments.

 
PMN concentration in both interstitium and BAL was negligible at 0 h. After 4 h, 33% of all pulmonary PMN were found in the interstitium. At the same time, PMN represented 91% of all cells in the BAL (Fig. 3B). After 24 h, the majority of PMN (78%) in the lung were found in the extravascular space. Note that PMN in BAL appear GR-1– as the GR-1 antibody injected 5 min before death remains confined to the vasculature. In control animals, saline inhalation induced a mild PMN accumulation in the pulmonary vasculature. No migration into the interstitium or into the alveolar air space was observed in these mice (data not shown).

Kinetics of transendothelial and transepithelial PMN migration. Interstitium and air space were free of PMN at 0 h. Transendothelial migration into the interstitial space started 1 h after LPS exposure and increased continuously. After 12 h, the majority of PMN in the lung were found extravascular (interstitium and air space) (Fig. 4). Until 2 h, the intravascular accumulation outpaced the neutrophil accumulation in the extravascular space so that the proportion of extravascular PMN did not change. By contrast, at 4 h the rate of accumulation of intravascular neutrophils slowed, and extravascular PMN accounted for 33% of all neutrophils in the lung.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 4. Time course of PMN trafficking. At 0, 1, 2, 4, 12, and 24 h after LPS exposure, PMN concentrations were assessed in the different lung compartments (vasculature, interstitium, BAL). Pulmonary circulation was flushed to remove nonadherent cells. Absolute PMN counts were calculated by total leukocyte counts (Trypan blue exclusion), and relative PMN concentrations were obtained by flow cytometry. Means ± SE of n = 4 time-course experiments are displayed. *Significant difference in cell count from baseline at t = 0. Insets: cytospin of BAL at t = 0 (a) and t = 24 h (b). BAL of untreated mice was dominated by alveolar macrophages. Twenty-four hours after LPS inhalation, the cell population changed to predominantly PMN.

 
The kinetics of neutrophil recruitment into the BAL was different in that no cells were found at 1 h and only a very small number (130,000 per mouse) at 2 h, after which time neutrophil numbers increased rapidly and then followed the number of interstitial neutrophils with a delay of ~2 h.

The appearance of PMN in the BAL was confirmed by cytospin. Cytospins of BAL were analyzed in untreated mice and in mice 24 h after LPS inhalation. The predominant cells in BAL of untreated mice were alveolar macrophages (Fig. 4, left inset). As expected, PMN dominated the cell population 24 h after LPS exposure (Fig. 4, right inset).

Lung permeability. Vascular leakage was determined to confirm lung injury in our LPS inhalation model. Six hours after LPS inhalation, Evans blue extravasation was significantly higher compared with saline inhalation (66.2 ± 6.2 vs. 29.3 ± 3.7 µg per g lung; P = 0.002) (Fig. 5).



View larger version (9K):
[in this window]
[in a new window]
  		Fig. 5. To confirm lung injury in response to LPS inhalation, we determined vascular permeability in the lung using the Evans blue extravasation technique. Six hours after LPS inhalation, Evans blue extravasation into the lung was significantly higher compared with the control group, which had received saline inhalation (66.2 ± 6.2 vs. 29.3 ± 3.7 µg Evans blue per g lung, P = 0.002). Data are presented as means ± SE of n = 4 animals in each group. *Significant difference from saline control.

 
PTx treatment. In some mice, 4 µg of PTx (41) were injected intravenously 30 min before LPS challenge. Lungs and BAL were harvested 14 h after LPS exposure, and anti-GR-1 was injected 5 min before death to distinguish between intravascular and interstitial PMN. LPS induced a significant PMN migration into all three lung compartments. Mice pretreated with PTx exhibited normal PMN sequestration into the pulmonary vasculature but showed reduced PMN migration into the lung interstitium (0.5 ± 0.1 x 106 vs. 2.7 ± 0.4 x 106; P < 0.01) (Fig. 6, A and B). Accordingly, almost no PMN were found in the alveolar air space at 14 h after LPS (0.3 ± 0.01 x 106 vs. 2.8 ± 0.3 x 106, P < 0.01) (Fig. 6B). The inhibitory effect of PTx was dose dependent as shown in Fig. 7. This suggests that G{alpha}i is involved at least in transendothelial migration from blood space to interstitium and possibly also in transepithelial migration into the alveolar air space.



View larger version (38K):
[in this window]
[in a new window]
  		Fig. 6. To block G{alpha}i-dependent signaling, we injected pertussis toxin (PTx) 30 min before LPS challenge. PMN in lungs and BAL were assessed 14 h after LPS exposure. Anti GR-1-antibody was injected 5 min before death. PMN were identified by their expression of CD45 and 7/4. GR-1 was utilized to distinguish intravascular (GR-1+) and interstitial (GR-1–) PMN. LPS induced a significant PMN recruitment to all 3 compartments of the lung. PTx pretreatment sharply reduced PMN migration into the interstitium and almost completely eliminated PMN recruitment into the alveolar air space. In contrast, PMN counts in the vascular compartment remained unaffected. A: representative FACS plot of n = 3 experiments. B: absolute PMN counts in the different compartments of the lung (open bars, control; solid bars, LPS; hatched bars, LPS + PTx). *Significant difference in PMN counts from the control. Data are presented as means ± SE of n = 3 experiments in each group. IV, intravascular; IS, interstitial.

 


View larger version (10K):
[in this window]
[in a new window]
  		Fig. 7. PTx induced a dose-dependent inhibition of PMN migration into BAL (A) and interstitial space (B). PTx was injected intravenously 30 min before LPS exposure. PMN in the different lung compartments were determined after 12 h. No effect of PTx on PMN recruitment into the pulmonary vasculature was observed (C).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In an LPS-induced model of acute lung injury, the sequential migration of PMN into the different compartments of the lung was explored. Using a new and quantitative flow cytometry-based technique, we show that LPS inhalation induced a rapid PMN sequestration in the pulmonary vasculature. Migration into the lung interstitium was observed within 1 h after LPS exposure, while transepithelial migration was delayed. PTx sharply reduced migration into the interstitium and into the alveolar air space but did not affect vascular accumulation.

Methodological considerations. When antibody injection is used to identify intravascular neutrophils in the lung, the antibody is required to reach all PMN in this compartment. We found a complete GR-1 labeling of all PMN in the systemic circulation. Additionally, the accessibility of the pulmonary vasculature was successfully tested using a marker for erythrocytes.

Even within 5 min, the GR-1 antibody might leak and stain extravascular PMN. In our studies, GR-1+ PMN did not appear until 4 h after antibody injection in the BAL, indicating that the antibody did not reach alveolar air space. To test for antibody leakage into the interstitium, we injected a higher (10-fold) dose of GR-1 that should increase the amount of leaked antibody and lead to an increased fraction of labeled PMN. No such increase was observed in our experiments (data not shown). Finally, a possible endothelial leakage should increase as the LPS-induced alveolo-capillary damage proceeds over time, leading to a continuously rising overestimation of the intravascular PMN fraction. However, intravascular PMN concentration peaks at 4 h after LPS, while PMN concentration in the interstitium increases at this time (Fig. 4). Together these data suggest that antibody leakage into the interstitium did not play a major role in our study.

Alveolar PMN are removed by BAL. Inaccessible airways might exist, particularly in the inflamed lung, leaving (GR-1–) PMN in the air space. This might result in an overestimation of the PMN counts in the interstitial compartment. However, significant contribution of alveolar PMN to the fraction of GR-1– PMN would be reflected by an either constant or increased ratio between interstitial and total extravascular (interstitial + alveolar) PMN over time. In fact, the fraction of interstitial PMN in all extravascular PMN decreases over time (Fig. 4). In addition, PMN counts in the interstitium reach a plateau after 12 h, while the PMN concentration in the air space still arises. Therefore, this effect does not significantly contribute to the results.

Investigating PMN migration in the lung. Although the lung offers a unique system to study cell migration, molecular mechanisms are still largely unknown.

Transendothelial migration from the vasculature into the lung interstitium was not measured in earlier studies. Indirect measurements, such as a drop in PMN blood counts or lung MPO activity, are not suitable to study this first important migration step. Current knowledge about the interaction between PMN and pulmonary endothelium derives mostly from in vitro studies using endothelial cell lines (25, 30). Attempts were recently made to mimic more realistically the alveolo-capillary barrier in an in vitro system (20). It remains to be shown whether this method will provide insight into pulmonary PMN migration.

Our flow cytometry-based approach is able to reflect many aspects of PMN trafficking in vivo, including the presence of a physiological marginated pool (9), accumulation after challenge, as well as the different migration steps into the alveolar air space and the LPS-induced release of PMN from the bone marrow (37, 46).

Kinetics of PMN trafficking. Several studies addressed the kinetics of PMN trafficking in the lung. Most of them focused on the initial retention in the pulmonary capillaries. Mathematical models (18, 21), multiple indicator techniques (38), injection of labeled PMN (17), as well as isolated lung models have been developed to describe PMN retention in the lung. PMN sequestration in the pulmonary vasculature in response to various inflammatory stimuli, such as live bacteria (13), complement fragments (31), macrophage inflammatory protein-2 (16), or LPS (2), occurs rapidly within a few minutes. PMN migration into the lung was detected as early as 1–2 h after injection of C5-fragment or Escherichia coli as assessed by radiolabeled PMN (11, 15). PMN infiltration into the BAL has been shown to be delayed for up to 6 h (12, 13, 43, 44). We found evidence of PMN migration into the alveolar air space starting after 2 h with a peak between 12 and 24 h. At 4 h, vascular PMN accumulation reached its maximum, indicating that PMN recruitment from the peripheral circulation was balanced by migration into the interstitium and the alveolar air space at an equal rate. The interstitial space held a significant number of PMN during the migration process, indicating that this space functions as a discrete compartment after an inflammatory stimulus.

Leukocyte-endothelial interactions are essential for the PMN recruitment to the lung (34). The engagement of molecules required for PMN recruitment, such as adhesion molecules and chemokines, varies among different inflammation models. There is good evidence that distinct signals are required for PMN to migrate through the different barriers, and even a single mediator can affect the migration steps differentially. For instance, nitric oxide induces vascular PMN sequestration in a murine model of sepsis but attenuates migration into the alveolar air space (35). In our study, PTx was able to block both transendothelial and transepithelial migration. However, the vascular accumulation was largely unaffected, indicating that chemokine receptor signaling is not required for neutrophil arrest in the pulmonary circulation. It has been previously suggested that chemokines and adhesion molecules both contribute to PMN arrest in the systemic microcirculation (41). It remains to be shown whether this mechanism applies in the lung as well.

The delay of transepithelial PMN migration in PTx-treated mice supports the hypothesis that a distinct signal is required for PMN to advance from the lung interstitium into the alveolar air space. Interestingly, PMN crossing the epithelial barrier seem to be pivotal for inducing lung damage associated with an increase in mortality (23).

Our data establish the first quantitative method for monitoring neutrophil migration from blood to lung interstitium to alveolar air space. Vascular sequestration occurred immediately after LPS challenge, whereas transendothelial and transepithelial migration into the air space was delayed. In ALI, the lung interstitium holds a significant amount of PMN during the migration process. Distinguishing intravascular and interstitial PMN in vivo facilitates new opportunities to study the regulation of PMN migration in the lung.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by Medical Faculty of the University of Tübingen Grant 1099-1-0 (to J. Reutershan) and by National Heart, Lung, and Blood Institute Grant HL-73361 (to J. Linden and K. Ley).


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Ley, Univ. of Virginia Health System, Cardiovascular Research Center, PO Box 801394, Charlottesville, VA 22908-1394 (e-mail: klausley{at}virginia.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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abraham E. Neutrophils and acute lung injury. Crit Care Med 31: S195–S199, 2003.[CrossRef][ISI][Medline]
  2. Andonegui G, Bonder CS, Green F, Mullaly SC, Zbytnuik L, Raharjo E, and Kubes P. Endothelium-derived Toll-like receptor-4 is the key molecule in LPS-induced neutrophil sequestration into lungs. J Clin Invest 111: 1011–1020, 2003.[CrossRef][ISI][Medline]
  3. Andonegui G, Goyert SM, and Kubes P. Lipopolysaccharide-induced leukocyte-endothelial cell interactions: a role for CD14 versus toll-like receptor 4 within microvessels. J Immunol 169: 2111–2119, 2002.[Abstract/Free Full Text]
  4. Belperio JA, Keane MP, Burdick MD, Londhe V, Xue YY, Li K, Phillips RJ, and Strieter RM. Critical role for CXCR2 and CXCR2 ligands during the pathogenesis of ventilator-induced lung injury. J Clin Invest 110: 1703–1716, 2002.[CrossRef][ISI][Medline]
  5. Burns AR, Smith CW, and Walker DC. Unique structural features that influence neutrophil emigration into the lung. Physiol Rev 83: 309–336, 2003.[Abstract/Free Full Text]
  6. Burns JA, Issekutz TB, Yagita H, and Issekutz AC. The beta2, alpha4, alpha5 integrins and selectins mediate chemotactic factor and endotoxin-enhanced neutrophil sequestration in the lung. Am J Pathol 158: 1809–1819, 2001.[Abstract/Free Full Text]
  7. Cotter MJ, Norman KE, Hellewell PG, and Ridger VC. A novel method for isolation of neutrophils from murine blood using negative immunomagnetic separation. Am J Pathol 159: 473–481, 2001.[Abstract/Free Full Text]
  8. Doerschuk CM. NO and neutrophils during sepsis: NO says "yes" to sequestration but "no" to migration. Am J Respir Crit Care Med 170: 205–206, 2004.[Free Full Text]
  9. Doerschuk CM, Allard MF, Martin BA, MacKenzie A, Autor AP, and Hogg JC. Marginated pool of neutrophils in rabbit lungs. J Appl Physiol 63: 1806–1815, 1987.[Abstract/Free Full Text]
  10. Doerschuk CM, Quinlan WM, Doyle NA, Bullard DC, Vestweber D, Jones ML, Takei F, Ward PA, and Beaudet AL. The role of P-selectin and ICAM-1 in acute lung injury as determined using blocking antibodies and mutant mice. J Immunol 157: 4609–4614, 1996.[Abstract]
  11. Doherty DE, Downey GP, Worthen GS, Haslett C, and Henson PM. Monocyte retention and migration in pulmonary inflammation. Requirement for neutrophils. Lab Invest 59: 200–213, 1988.[ISI]
  12. Downey GP, Worthen GS, Henson PM, and Hyde DM. Neutrophil sequestration and migration in localized pulmonary inflammation. Capillary localization and migration across the interalveolar septum. Am Rev Respir Dis 147: 168–176, 1993.[ISI][Medline]
  13. Doyle NA, Bhagwan SD, Meek BB, Kutkoski GJ, Steeber DA, Tedder TF, and Doerschuk CM. Neutrophil margination, sequestration, and emigration in the lungs of L-selectin-deficient mice. J Clin Invest 99: 526–533, 1997.[ISI][Medline]
  14. Fan J and Malik AB. Toll-like receptor-4 (TLR4) signaling augments chemokine-induced neutrophil migration by modulating cell surface expression of chemokine receptors. Nat Med 9: 315–321, 2003.[CrossRef][ISI][Medline]
  15. Gao X, Xu N, Sekosan M, Mehta D, Ma SY, Rahman A, and Malik AB. Differential role of CD18 integrins in mediating lung neutrophil sequestration and increased microvascular permeability induced by Escherichia coli in mice. J Immunol 167: 2895–2901, 2001.[Abstract/Free Full Text]
  16. Gupta S, Feng L, Yoshimura T, Redick J, Fu SM, and Rose CE Jr. Intra-alveolar macrophage-inflammatory peptide 2 induces rapid neutrophil localization in the lung. Am J Respir Cell Mol Biol 15: 656–663, 1996.[Abstract]
  17. Han L, Saito H, Fukatsu K, Inoue T, Yasuhara H, Furukawa S, Matsuda T, Lin MT, and Ikeda S. Ex vivo fluorescence microscopy provides simple and accurate assessment of neutrophil-endothelial adhesion in the rat lung. Shock 16: 143–147, 2001.[ISI][Medline]
  18. Hanger CC, Wagner WW Jr, Janke SJ, Lloyd TC Jr, and Capen RL. Computer simulation of neutrophil transit through the pulmonary capillary bed. J Appl Physiol 74: 1647–1652, 1993.[Abstract/Free Full Text]
  19. Henderson RB, Hobbs JAR, Mathies M, and Hogg N. Rapid recruitment of inflammatory monocytes is independent of neutrophil migration. Blood 102: 328–335, 2003.[Abstract/Free Full Text]
  20. Hermanns MI, Unger RE, Kehe K, Peters K, and Kirkpatrick CJ. Lung epithelial cell lines in coculture with human pulmonary microvascular endothelial cells: development of an alveolo-capillary barrier in vitro. Lab Invest 84: 736–752, 2004.[CrossRef][ISI][Medline]
  21. Huang Y, Doerschuk CM, and Kamm RD. Computational modeling of RBC and neutrophil transit through the pulmonary capillaries. J Appl Physiol 90: 545–564, 2001.[Abstract/Free Full Text]
  22. Kina T, Ikuta K, Takayama E, Wada K, Majumdar AS, Weissman IL, and Katsura Y. The monoclonal antibody TER-119 recognizes a molecule associated with glycophorin A and specifically marks the late stages of murine erythroid lineage. Br J Haematol 109: 280–287, 2000.[CrossRef][ISI][Medline]
  23. Li Q, Park PW, Wilson CL, and Parks WC. Matrilysin shedding of syndecan-1 regulates chemokine mobilization and transepithelial efflux of neutrophils in acute lung injury. Cell 111: 635–646, 2002.[CrossRef][ISI][Medline]
  24. Lorenz E, Jones M, Wohlford-Lenane C, Meyer N, Frees KL, Arbour NC, and Schwartz DA. Genes other than TLR4 are involved in the response to inhaled LPS. Am J Physiol Lung Cell Mol Physiol 281: L1106–L1114, 2001.[Abstract/Free Full Text]
  25. Mackarel AJ, Russell KJ, Ryan CM, Hislip SJ, Rendall JC, FitzGerald MX, and O'Connor CM. CD18 dependency of transendothelial neutrophil migration differs during acute pulmonary inflammation. J Immunol 167: 2839–2846, 2001.[Abstract/Free Full Text]
  26. Markowicz P, Wolff M, Djedaini K, Cohen Y, Chastre J, Delclaux C, Merrer J, Herman B, Veber B, Fontaine A, and Dreyfuss D. Multicenter prospective study of ventilator-associated pneumonia during acute respiratory distress syndrome. Incidence, prognosis, and risk factors. ARDS study group. Am J Respir Crit Care Med 161: 1942–1948, 2000.[Abstract/Free Full Text]
  27. Matthay MA, Zimmerman GA, Esmon C, Bhattacharya J, Coller B, Doerschuk CM, Floros J, Gimbrone MA Jr, Hoffman E, Hubmayr RD, Leppert M, Matalon S, Munford R, Parsons P, Slutsky AS, Tracey KJ, Ward P, Gail DB, and Harabin AL. Future research directions in acute lung injury: summary of a National Heart, Lung, and Blood Institute working group. Am J Respir Crit Care Med 167: 1027–1035, 2003.[Abstract/Free Full Text]
  28. Maus UA, Waelsch K, Kuziel WA, Delbeck T, Mack M, Blackwell TS, Christman JW, Schlondorff D, Seeger W, and Lohmeyer J. Monocytes are potent facilitators of alveolar neutrophil emigration during lung inflammation: role of the CCL2-CCR2 axis. J Immunol 170: 3273–3278, 2003.[Abstract/Free Full Text]
  29. Miller EJ, Cohen AB, Nagao S, Griffith D, Maunder RJ, Martin TR, Weiner-Kronish JP, Sticherling M, Christophers E, and Matthay MA. Elevated levels of NAP-1/interleukin-8 are present in the airspaces of patients with the adult respiratory distress syndrome and are associated with increased mortality. Am Rev Respir Dis 146: 427–432, 1992.[ISI][Medline]
  30. Moreland JG, Bailey G, Nauseef WM, and Weiss JP. Organism-specific neutrophil-endothelial cell interactions in response to Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus. J Immunol 172: 426–432, 2004.[Abstract/Free Full Text]
  31. Olson TS, Singbartl K, and Ley K. L-selectin is required for fMLP- but not C5a-induced margination of neutrophils in pulmonary circulation. Am J Physiol Regul Integr Comp Physiol 282: R1245–R1252, 2002.[Abstract/Free Full Text]
  32. Peng X, Hassoun PM, Sammani S, McVerry BJ, Burne MJ, Rabb H, Pearse D, Tuder RM, and Garcia JGN. Protective effects of sphingosine 1-phosphate in murine endotoxin-induced inflammatory lung injury. Am J Respir Crit Care Med 169: 1245–1251, 2004.[Abstract/Free Full Text]
  33. Pruijt JFM, Verzaal P, van Os R, de Kruijf EJ, van Schie MLJ, Mantovani A, Vecchi A, Lindley IJD, Willemze R, Starckx S, Opdenakker G, and Fibbe WE. Neutrophils are indispensable for hematopoietic stem cell mobilization induced by interleukin-8 in mice. Proc Natl Acad Sci USA 99: 6228–6233, 2002.[Abstract/Free Full Text]
  34. Puneet P, Moochhala S, and Bhatia M. Chemokines in acute respiratory distress syndrome. Am J Physiol Lung Cell Mol Physiol 288: L3–L15, 2005.[Abstract/Free Full Text]
  35. Razavi HM, Wang LF, Weicker S, Rohan M, Law C, McCormack DG, and Mehta S. Pulmonary neutrophil infiltration in murine sepsis: role of inducible nitric oxide synthase. Am J Respir Crit Care Med 170: 227–233, 2004.[Abstract/Free Full Text]
  36. Reutershan J and Ley K. Bench-to-bedside review: acute respiratory distress syndrome - how neutrophils migrate into the lung. Crit Care 8: 453–461, 2004.[CrossRef][ISI][Medline]
  37. Sato Y, Van Eeden SF, English D, and Hogg JC. Bacteremic pneumococcal pneumonia: bone marrow release and pulmonary sequestration of neutrophils. Crit Care Med 26: 501–509, 1998.[CrossRef][ISI][Medline]
  38. Schwab AJ, Salamand A, Merhi Y, Simard A, and Dupuis J. Kinetic analysis of pulmonary neutrophil retention in vivo using the multiple-indicator-dilution technique. J Appl Physiol 95: 279–291, 2003.[Abstract/Free Full Text]
  39. Sikora L, Johansson AC, Rao SP, Hughes GK, Broide DH, and Sriramarao P. A murine model to study leukocyte rolling and intravascular trafficking in lung microvessels. Am J Pathol 162: 2019–2028, 2003.[Abstract/Free Full Text]
  40. Skerrett SJ, Liggitt HD, Hajjar AM, Ernst RK, Miller SI, and Wilson CB. Respiratory epithelial cells regulate lung inflammation in response to inhaled endotoxin. Am J Physiol Lung Cell Mol Physiol 287: L143–L152, 2004.[Abstract/Free Full Text]
  41. Smith ML, Olson TS, and Ley K. CXCR2- and E-selectin-induced neutrophil arrest during inflammation in vivo. J Exp Med 200: 935–939, 2004.[Abstract/Free Full Text]
  42. Sue RD, Belperio JA, Burdick MD, Murray LA, Xue YY, Dy MC, Kwon JJ, Keane MP, and Strieter RM. CXCR2 is critical to hyperoxia-induced lung injury. J Immunol 172: 3860–3868, 2004.[Abstract/Free Full Text]
  43. Tasaka S, Qin L, Saijo A, Albelda SM, DeLisser HM, and Doerschuk CM. Platelet endothelial cell adhesion molecule-1 in neutrophil emigration during acute bacterial pneumonia in mice and rats. Am J Respir Crit Care Med 167: 164–170, 2003.[Abstract/Free Full Text]
  44. Wagner JG, Harkema JR, and Roth RA. Pulmonary leukostasis and the inhibition of airway neutrophil recruitment are early events in the endotoxemic rat. Shock 17: 151–158, 2002.[CrossRef][ISI][Medline]
  45. Wang LF, Patel M, Razavi HM, Weicker S, Joseph MG, McCormack DG, and Mehta S. Role of inducible nitric oxide synthase in pulmonary microvascular protein leak in murine sepsis. Am J Respir Crit Care Med 165: 1634–1639, 2002.[Abstract/Free Full Text]
  46. Yamada M, Kubo H, Kobayashi S, Ishizawa K, Numasaki M, Ueda S, Suzuki T, and Sasaki H. Bone marrow-derived progenitor cells are important for lung repair after lipopolysaccharide-induced lung injury. J Immunol 172: 1266–1272, 2004.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
A. Zarbock, T. L. Deem, T. L. Burcin, and K. Ley
G{alpha}i2 is required for chemokine-induced neutrophil arrest
Blood, November 15, 2007; 110(10): 3773 - 3779.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
J. Reutershan, R. Stockton, A. Zarbock, G. W. Sullivan, D. Chang, D. Scott, M. A. Schwartz, and K. Ley
Blocking p21-activated Kinase Reduces Lipopolysaccharide-induced Acute Lung Injury by Preventing Polymorphonuclear Leukocyte Infiltration
Am. J. Respir. Crit. Care Med., May 15, 2007; 175(10): 1027 - 1035.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
A. D. Gregory, L. A. Hogue, T. W. Ferkol, and D. C. Link
Regulation of systemic and local neutrophil responses by G-CSF during pulmonary Pseudomonas aeruginosa infection
Blood, April 15, 2007; 109(8): 3235 - 3243.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
S. Perkowski, A. Scherpereel, J.-C. Murciano, E. Arguiri, C. C. Solomides, S. M. Albelda, V. Muzykantov, and M. Christofidou-Solomidou
Dissociation between alveolar transmigration of neutrophils and lung injury in hyperoxia
Am J Physiol Lung Cell Mol Physiol, November 1, 2006; 291(5): L1050 - L1058.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
P. Kinniry, Y. Amrani, A. Vachani, C. C. Solomides, E. Arguiri, A. Workman, J. Carter, and M. Christofidou-Solomidou
Dietary Flaxseed Supplementation Ameliorates Inflammation and Oxidative Tissue Damage in Experimental Models of Acute Lung Injury in Mice
J. Nutr., June 1, 2006; 136(6): 1545 - 1551.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/5/L807    most recent
00477.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (20)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Reutershan, J.
Right arrow Articles by Ley, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Reutershan, J.
Right arrow Articles by Ley, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.