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LPS from bacteria is ubiquitous in the environment and can cause airway disease and modify allergic asthma. Identification of gene products that modulate the biologic response to inhaled LPS will improve our understanding of inflammatory airways disease. Previous work has identified quantitative trait loci for the response to inhaled LPS on chromosomes 2 and 11. In these regions, 28 genes had altered RNA expression after inhalation of LPS, including CD44, which was associated with differences in both TNF-α levels and neutrophil recruitment into the lung. It has previously been shown that CD44 can modulate macrophage recruitment in response to Mycobacterium tuberculosis, as well as clearance of neutrophils after lung injury with both bleomycin and live Escherichia coli bacteria. In this study, we demonstrate that the biologic response to inhaled LPS is modified by CD44. Macrophages failed to be recruited to the lungs of CD44-deficient animals at all time points after LPS exposure. CD44-deficient macrophages showed reduced motility in a Transwell migration assay, reduced ability to secrete the proinflammatory cytokine TNF-α, reduced in vivo migration in response to monocyte chemotactic protein-1, and diminished adhesion to vascular endothelia in the presence of TNF-α. In addition, CD44-deficient animals had 150% fewer neutrophils at 24 h and 50% greater neutrophils 48 h after LPS exposure. These results support the role of CD44 in modulating the biologic response to inhaled LPS.
CD44 regulates macrophage accumulation in the lungs of mice exposed to inhaled endotoxin. CD44-dependent defects in macrophage migration are, in part, due to reduced endothelium adhesion. CD44 plays an important role in pulmonary innate immunity.
Inhalation of bacterial LPS can cause severe inflammatory disease in the lung. The initial host response to inhaled LPS includes acute neutrophil inflammation with increased expression of proinflammatory cytokines, followed by the recruitment of alveolar macrophages, which play a role in clearance of apoptotic neutrophils. While the acute proinflammatory response facilitates efficient clearance of inhaled environmental agents and pathogens, it can also lead to tissue injury and compromised lung function when not adequately regulated (1). Regulation of the inflammatory response is therefore necessary for effective elimination of bacterial pathogens and inhaled toxins, and resolution of tissue injury. Accordingly, it is of considerable importance to identify the molecular mechanisms that initiate inflammatory cell recruitment and clearance from the lung.
The biologic response to inhaled LPS is initiated by the receptor complex that includes Toll-like receptor 4 (TLR4), CD14, and MD-2. It is clear however, that other, uncharacterized genes play a role in the biologic response to inhaled LPS (2). Our laboratory recently performed a genome-wide quantitative trait locus (QTL) analysis and microarray-based gene expression study to identify novel candidate genes responsible for regulating the biologic response to inhaled LPS (3). In that study, ~ 500 known genes and expressed sequence tags (ESTs) were identified with significantly changed expression after inhalation of LPS. In addition, QTLs on chromosomes 2 and 11 affecting PMN recruitment and TNF-α protein production in the lung were identified. Of the 500 genes, 28 were found to be within chromosomal regions highlighted by the identified QTLs. One of the genes identified on the chromosome 2 QTL affecting TNF-α production was a previously uncharacterized EST, which represents an isoform of CD44 (GenBank BG063885). In that study, CD44 expression was associated with differences in TNF-α levels and modified neutrophil recruitment between resistant and susceptible strains. These associations suggested that CD44 might play a role in both proinflammatory cytokine production and neutrophil recruitment to the lung.
CD44 is a major cell-surface type I transmembrane receptor for hyaluronan (hyaluronic acid [HA]) and osteopontin that is expressed by most cells types, including; fibroblasts, neurons, leukocytes, and myeloid cells. HA assumes a significant role in maintaining tissue integrity as a ubiquitous element of the extracellular matrix. Activation of CD44 has previously been shown to regulate the inflammatory response. For example, ligation of CD44 with short fragment HA can induce a number of proinflammatory cytokines (4), CD44-deficient mice show increased mortality and unremitting inflammation in a mouse model of bleomycin-induced lung injury (5), and CD44 plays a role in both migration into and clearance of neutrophils from the lungs in response to Escherichia coli pneumonia in mice (6). CD44 also directly regulates neutrophil migration in that CD44-deficient cells demonstrate impaired migration in vitro (7). CD44 interaction with HA can also regulate in vitro lymphocyte adhesion (8). Studies in infectious and noninfectious models of lung injury suggest that CD44 can modify both inflammatory cell recruitment to the lung via ligation to HA (6) and clearance of apoptotic neutrophils by macrophages during the resolution of lung injury (5).
Given these genetic and biologic considerations, we hypothesized that CD44-deficient mice would have enhanced inflammation after exposure to inhaled LPS as a result of impaired clearance. To address this hypothesis, we exposed C57BL/6J and CD44-deficient animals to aerosolized LPS. In this study, we show that CD44 plays a critical role in the recruitment of macrophages into the lung in response to inhaled LPS. We also demonstrate the role of CD44 in macrophage adhesion to endothelia and macrophage chemotaxis after either inhalation of LPS or exposure to the chemokine, monocyte chemotactic protein (MCP)-1.
Genetically engineered CD44-deficient mice were generously provided by Dr. Tak Mak from the University Health Network in Toronto, Canada (9), and were backcrossed onto a C57BL/6J background for eight generations. Each experimental group consists of 5–10 male mice at 6–8 wk of age. Experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Duke University Medical Center and performed in accordance with the standards established by the U.S. Animal Welfare Acts.
LPS was purchased as lyophilized, purified 0111:B4 E. coli (Sigma, St. Louis, MO). LPS aerosol was generated and monitored as previously described (10). The dosage is similar to that experienced by grain mill workers during a typical 8-h work day and causes an inflammatory response in the lower respiratory tract. The target concentration of LPS aerosol generated in these experiments was 4–6 μg/m3.
Recombinant murine MCP-1 was purchased from R&D Systems (Minneapolis, MN). Mice were anesthetized with 3% isoflurane, and 12.5 μg MCP-1 in 50 μl sterile saline was instilled by oropharyngeal aspiration (11). Animals were phenotyped 24 h after exposure.
Whole lung lavage was performed as previously described (10). Lavage cells were spun onto a glass slide using a cytocentrifuge (Cytospin-2; Shanden Southern, Sewickley, PA) and stained using a Diff Quick Stain Set (Harleco, Gibbstown, NY). Cell-free lavage supernatants were stored at −70°C.
A Luminex instrument (Bio-Rad, Hercules, CA) was used to evaluate protein concentrations of IL-1β, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), TNF-α, and macrophage inflammatory protein (MIP)-1α (Bio-Rad). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, MIP-2, IL-6, and keratinocyte cytokines (KC) were purchased from R&D Systems. Hyaluronan ELISA kit was purchased from Echelon Biosciences Inc. (Salt Lake City, UT). Reagents were used according to the manufacturer's instructions, with the exception of using an overnight incubation with Luminex assays. The lower limit of detection for each protein was 6 pg/ml for TNF-α, 7.0 pg/ml for IL-1β, 1.5 pg/ml for MIP-2, 1.6 pg/ml for IL-6, 1 pg/ml for G-CSF, 7 pg/ml for GM-CSF, 24 pg/ml for MIP-1α, and 2.0 pg/ml for KC.
Thioglycolate elicited peritoneal macrophages were obtained from C57BL/6J mice and CD44-deficient mice by intraperitoneal injection of 2 ml of 4% Brewer's thioglycolate and harvesting of cells 72 h later. Peritoneal macrophages were pooled from three animals, washed, and resuspended in RPMI with 10% heat inactivated fetal calf serum (FCS) and 1% streptomycin/1% penicillin. Five wells per group were plated on 24-well culture plates at 1 × 106 cells/ml. We challenged these cells to increasing doses of 0111:B4 E. coli LPS (Sigma) for 4 h.
Carboxy-fluorescein diacetate, succinimidyl ester (CFSE)-labeled peritoneal macrophages and bone marrow–derived neutrophils were used for migration and adhesion assays. Peritoneal macrophages were obtained as described above. Neutrophils were isolated from murine bone marrow by Percoll gradient centrifugation (12). Briefly, femurs and tibias of C57BL/6 and CD44−/− mice were dissected, the marrows were flushed out with Hanks' buffered saline solution (HBSS; GIBCO BRL, Gaithersburg, MD) and passed through a syringe with a 20-gauge needle several times. Separated single bone marrow cells were then layered on a three-step Percoll (Pharmacia, Piscataway, NJ) gradient (52%/64%/72%), which was centrifuged at 1,060 × g for 25 min. From the interface between the 64% and 72% fractions, neutrophils were collected and washed twice. Isolated macrophages and neutrophils were then resuspended in PBS with 0.1% BSA and incubated with 50 μM and 5 μM of CFSE (Molecular Probes, Carlsbad, CA), respectively, for 10 min at 37°C. Staining was quenched with 20% FCS. After washing off CFSE in medium, cells labeled with CFSE fluorescence could be seen under a fluorescent microscope. For adhesion assay, human umbilical vein endothelial cells (HUVEC; Cell Applications Inc. San Diego, CA.) were cultured in clear black 96-well plate to 100% confluence and either pretreated with or without TNF-α (20 ng/ml) or LPS (1 μg/ml) for 4 h. A quantity of 1 × 105 of CFSE-labeled macrophages or 2.5 × 105 of CFSE-labeled neutrophils was added to HUVEC layer and co-cultured for 30 and 60 min at 37°C in a 5% CO2 atmosphere. Nonadherent cells were washed off with HBSS. CFSE fluorescence was measured at Ex 485nm and Em 535nm using a DTX800 Multimode Detector from Beckman Coulter, Inc. (Fullerton, CA). Adherent cells were expressed as percentage of total loaded cells.
For macrophage migration assay, 24 Transwell plates with 5 μm pore size of membrane were used. A quantity of 1 × 105 of CFSE-labeled macrophages in FCS-free DMEM was loaded to the upper well, and 600 μl of saline with or without chemoattractants (M-CSF, 20 ng/ml; MCP-1, 200 ng/ml; fMLP, 100 nM) or 1:2 diluted lavage supernatants from LPS exposed wild-type or CD44−/− mice were loaded to the lower well. After 20 h incubation at 37°C with 5% CO2, cells attached to both sides of the membrane were washed twice with HBSS. Cotton swabs were used to remove cells on either the upper or lower side of the membrane. Under a fluorescence microscope, migrating cells on the lower side of the membrane were counted in five high-power fields. For neutrophil migration assay, Transwell plates with same pore size of membrane were used. A quantity of 1 × 106 of neutrophils in RPMI 1640 with 10% FCS were loaded to upper well and incubated with 600 μl of testing medium (LTB4 100 nM) in lower well for 1 h at 37°C with 5% CO2. Cells were collected from both sides of each wells and counted using a hemocytometer. Migrating neutrophils were expressed as percentage of total loaded cells. Above migration assays were repeated twice and each group contained three Transwells.
Data are expressed as mean ± SEM. Significant differences between groups were identified by two-way ANOVA and individual comparisons were made using a Student t test. A two-tailed P value of < 0.05 was considered statistically significant.
To characterize the role of CD44 in the initiation, progression, and clearance of cellular inflammation, we examined animals at multiple time points after a single LPS inhalation challenge. In vivo exposures were performed at levels comparable to occupational exposures to inhaled LPS by agricultural workers (13). At baseline, the concentration of macrophages in the lungs were not significantly different between C57BL/6 and CD44-deficient (C57BL/6CD44−/−) mice (WT 25,944 ± 7,084 versus CD44−/− 18,521 ± 2,861 cells/ml lavage fluid, n = 9; P = 0.28). Four hours after a single acute exposure to inhaled LPS, there was no difference between C57BL/6 mice and C57BL/6CD44−/− mice in total inflammatory cells in whole lung lavage fluid (Figure 1). Interestingly, 24 h after LPS inhalation challenge, C57BL/6 CD44−/− mice had significantly fewer total inflammatory cells and neutrophils when compared with wild-type animals (Figure 1). However, there were marked differences in the concentration of lavage macrophages with significantly more macrophages in the lower airspace of C57BL/6 mice compared with C57BL/6CD44−/− mice between 4 and 96 h after inhalation of LPS (Figure 1). Moreover, there was no difference between the concentration of macrophages in LPS-exposed C57BL/6CD44−/− mice and unexposed C57BL/6CD44−/− mice, supporting the idea that macrophage accumulation in the airspace was attenuated in C57BL/6CD44−/− mice after exposure to LPS. Forty-eight hours after the LPS exposure, the number of inflammatory cells in whole lung lavage fluid was not different between the groups. However, the C57BL/6CD44−/− mice continued to have significantly fewer macrophages compared with C57BL/6 mice up to 96 h after the LPS exposure (Figure 1), and interestingly, had significantly more neutrophils at 48 h, when compared with C57BL/6 mice. Thus CD44 deficiency is associated with reduced macrophage accumulation in the lower airspace after exposure to inhaled LPS.
Because of the failure to recruit macrophages to the lung in response to a single LPS inhalation challenge, we reasoned that repeated LPS inhalation challenge such as an agricultural worker might be exposed to in the course of a work week would result in significantly elevated neutrophil inflammation in C57BL/6CD44−/− mice due to reduced macrophage accumulation and therefore clearance of apoptotic neutrophils. This would be consistent with a recent report that bleomycin-induced lung injury in CD44-deficient mice is associated with impaired clearance of neutrophils from the lung and that C57BL/6CD44−/− mice might be more susceptible to enhanced lung injury from repeated LPS exposures. To address this question, we exposed C57BL/6 mice and C57BL/6CD44−/− mice to five consecutive days of aerosolized LPS inhalation challenge. Interestingly, there was no significant difference in cellular inflammation in the lungs between these two groups after multiple days of aerosol exposure to inhaled LPS (data not shown).
To determine whether the impaired recruitment of macrophages in C57BL/6CD44−/− mice after a single LPS inhalation challenge was due to a primary defect in CD44-dependent macrophage function, we exposed peritoneal macrophages from C57BL/6 and C57BL/6CD44−/− mice to LPS in vitro. CD44-deficient macrophages produced significantly less TNF-α in response to increasing doses of LPS when compared with C57BL/6 wild-type macrophages (Figure 2). These in vitro results suggest that CD44 can directly modify the biologic response to LPS in macrophages.
Because we observed impaired recruitment of macrophages after a single LPS inhalation exposure in C57BL/6CD44−/− mice and because peritoneal macrophages from C57BL/6CD44−/− mice produced less TNF-α in vitro, we asked whether there was a difference in inflammatory mediators in whole lung lavage between C57BL/6 and C57BL/6CD44−/− mice after LPS inhalation. Surprisingly, there were no differences in TNF-α, IL-1α, GM-CSF, MIP-1α, or KC protein contents in whole lung lavage fluid between groups. However, immediately after exposure to endotoxin there was a significant increase in C57BL/6J versus C57BL/6CD44−/− mice in secreted factors, including IL-1β (89 ± 4 pg/ml versus 57 ± 5 pg/ml; P = 0.002), IL-6 (154 ± 8 pg/ml versus 121 ± 5 pg/ml; P = 0.03), and G-CSF (391 ± 23 pg/ml versus 302 ± 22 pg/ml; P = 0.01). At early time points, there was a trend toward higher level of MIP-2 in C57BL/6J versus C57BL/6CD44−/− mice (610 ± 52 pg/ml versus 530 ± 71 pg/ml). By 24 h after exposure, MIP-2 production remained detectible in C57BL/6 mice (1.9 ± 0.4 pg/ml), while there was no detectable MIP-2 in whole lung lavage fluid from C57BL/6CD44−/− mice.
Previous studies suggest that CD44 can play a role in clearance of HA after lung injury. In this study, we also observed differences in the HA content of lavage fluid at several time points, though these were smaller than those previously reported (5). C57BL/6CD44−/− mice had 4-fold increase in HA in whole lung lavage at 4 h compared with C57BL/6 mice (24.2 ± 4.2 ng/ml versus 6.0 ± 0.6 ng/ml; P < 0.05). Twenty-four hours after LPS exposure, there were no detectible differences in HA concentrations in CD44-deficient animals. Forty-eight hours after the LPS exposure, C57BL/6CD44−/− mice had a 5-fold increase in HA in the lung lavage when compared with C57BL/6 mice (10.6 ± 2.1 ng/ml versus 2.1 ± 0.4 ng/ml; P < 0.05) at the same time point.
Alterations in the kinetics of lung inflammation after exposure to LPS could be the result of alterations in secreted factors, impaired chemotaxis, or impaired cellular adhesion. To determine the role of CD44-dependent secreted factors into the alveolar compartment, we confirmed that both wild-type and CD44-deficient PMNs maintain their ability to migrate to LTB4 and supernatants from lavage fluid obtained from either LPS-exposed C57BL/6 or C57BL/6CD44−/− mice (Figure 3). We did not observe CD44-dependent defects in neutrophil migration in this assay in the absence of HA matrix. We then investigated the importance of CD44 for the migration of macrophages. Peritoneal macrophages pre-attached to Transwell membranes were exposed to media, fMLP, GM-CSF, or MCP-1. After 21 h incubation, the concentration of migrated CD44-deficient macrophages was significantly less than that of migrated C57BL/6 macrophages under all conditions (Figure 3). In addition, we observed reduced migration of CD44-deficient peritoneal macrophages toward lavage supernatant obtained from either LPS-exposed C57BL/6 or C57BL/6CD44−/− mice (Figure 3).
To determine whether the CD44-dependent defect in monocyte recruitment into the lung is specific to LPS, we challenged animals with the chemokine MCP-1 as previously reported (11). CD44-deficient animals demonstrate a profound defect in monocyte recruitment into the lung after exposure to MCP-1 (Figure 4). After exposure to MCP-1, no differences in absolute numbers of either neutrophils or lymphocytes were observed dependent on CD44. This observation suggests that the CD44-dependent defect in monocyte recruitment is less likely a direct result of response to LPS. In contrast, this observation suggests a CD44-dependent defect in either secondary response to secreted factors or primary monocyte function.
Altered macrophage chemotaxis could result from either defective adhesion or altered diapedesis in response to chemoattractant. We considered whether macrophages demonstrate CD44-dependent adhesion as had previously described with endothelial cell adhesion to extracellular matrix (14). To address this issue, we preformed an in vitro macrophage–endothelial adhesion assay. In the absence of stimulation, CD44-deficient macrophages had significantly attenuated adhesion to endothelial cells (Figure 5). This difference was accentuated in the presence of TNF-α stimulation (Figure 5). In contrast, in the presence of TNF-α or LPS, we did not observe any CD44-dependent differences in neutrophil–endothelial adhesion (Figure 5). To better understand the mechanism of altered macrophage response to stimulation, we preformed in vitro studies to determine whether CD44 could modify the expression of either tlr4 or adhesion molecules. Bone marrow–derived macrophages from C57BL/6J or CD44−/− mice were stimulated with either LPS, TNF-α, short fragment HA, or long fragment HA. In this assay, the presence of CD44 did not modify the expression of either tlr4 or adhesion molecules, including CD47, CD54, CD18, CD11a, CD11b, and CD11c (flow cytometry data not shown). These results support a fundamental role of CD44 in macrophage–endothelial adhesion independent of tlr4 mRNA expression, which is enhanced in the presence of TNF-α.
Our results demonstrate that after a single exposure to clinically relevant concentrations of inhaled LPS, C57BL/6CD44−/− mice have significantly less macrophage recruitment to the lung. This defect in macrophage recruitment was associated with impaired in vitro production of TNF-α, altered migration in response to multiple chemoattractants, and reduced adhesion to endothelium. Taken together, these results show that recruitment of macrophages to the lung after exposure to inhaled LPS is, in part, dependent on CD44.
Primary defects in CD44-deficient macrophage migration to the lungs have been previously described following intranasal infection with Mycobacterium tuberculosis (15). In that study, CD44-deficient mice had a 50% reduction in pulmonary macrophages 2 wk after infection, suggesting that CD44 mediates resistance to mycobacterial infection through recruitment of macrophages at the site of infection and phagocytosis of the organism (15). Similarly, we demonstrate that 48 h after LPS inhalation the absolute number of total leukocytes remained unchanged between the groups, yet there was an overall defect in macrophage recruitment in C57BL/6CD44−/− mice. While our study does not address live infection with E. coli, our observations suggest that a defect in macrophage recruitment in response to E. coli LPS could have a substantial impact on inflammatory lung injury associated with live pathogens. It is likely that a combination of surface receptors regulate both the recognition and clearance of live pathogens.
Our study demonstrates a CD44-dependent defect in both in vitro production of TNF-α and macrophage recruitment to the lung. Our in vitro observation that the macrophage cytokine response to LPS is minimally attenuated in CD44-deficient animals suggests that CD44 can directly modify tlr4-dependent responses. We speculate that a complex interaction between CD44 signaling and tlr4 signaling is required for the complete biologic response to inhaled LPS, as has been previously suggested with both bleomycin lung injury (16) and tumor cell signaling (17). Similarly, previous studies have shown that recognition of M. tuberculosis is dependent on CD44 (15), nucleotide-binding oligomerization domain 2 (NOD2), and TLRs (18). It remains plausible that the initial LPS response is dependent on tlr4, while secondary signaling can be modified by CD44. In this study, we show elevated HA in lung lavage fluid in LPS-exposed CD44-deficient animals, supporting the role of CD44 in clearance of soluble HA. Increased level of HA in the lung has previously been associated with recruitment of macrophages into the lung after bleomycin-induced lung injury (19). Furthermore, there is evidence in the literature suggesting that hyaluronan can act as an endogenous ligand of tlr4 (16, 20, 21). We speculate that elevated levels of HA could modify local tlr4-dependent signaling in response to LPS. In addition, previous studies demonstrate that high-molecular-weight hyaluronan can attenuate inflammatory response (4, 22). It remains unclear whether the fragment size of HA can regulate the biologic consequences of HA ligation to TLRs. However, in this study, we demonstrate that CD44-dependent recruitment of macrophages to the lung after inhaled LPS is primarily related to defects in macrophage migration and endothelial adhesion.
Reduced absolute numbers of macrophages in the lung were associated with alterations in the kinetics of neutrophil trafficking after inhaled LPS. In this study, CD44-deficient animals had reduced concentration of PMNs at 24 h and increased numbers of PMNs at 48 h. Reduced concentration of neutrophils in CD44-deficient mice 24 h after exposure could be explained by several mechanisms, including a reduction in regulatory macrophages recruited into the lung, impaired PMN migration (6, 7), reduced endothelial adhesion (23), or reduced concentration of local chemokines. In this study, we did not observe biologically meaningful differences in either chemokines in lavage fluid or neutrophil migration in the absence of hyaluronan matrix in vitro. However, alveolar macrophages, which are known to play a critical role in neutrophil recruitment and clearance after exposure to inhaled LPS (24, 25), were reduced. We speculate that an absolute reduction in alveolar macrophages contributes to the reduced numbers of neutrophils at this time point. In addition, CD44 is known to play a role in the clearance of apoptotic neutrophils (5). However, in this study, we did not detect any differences in percentage of apoptotic neutrophils by flow cytometry and annexin V staining at 24 h (data not shown). In context of previous studies, our observations support that both the intensity and mechanism of lung injury dictate the modifying role of CD44. Recent work by Liang and coworkers supports the idea that when mice are exposed to very high doses of LPS associated with substantial lung injury, CD44 can act as a negative regulator of acute pulmonary inflammation (26). These observations support divergent roles of CD44 depending on the severity of lung injury.
The role of CD44 in lung inflammation appears dependent on the exposure as well as the duration, intensity, and timing after environmental challenge. While our results demonstrate that macrophage recruitment to the lung after exposure to low levels of aerosolized LPS is dependent on intact CD44, while the production of proinflammatory cytokines by macrophages is only partially dependent on CD44. Importantly, our data support a primary defect in macrophage–endothelial adhesion as a mechanism to explain an absolute reduction in the numbers of macrophages recruited into the lung. Defects in endothelial adhesion were associated with reduced recruitment of macrophages after single exposure to both low levels of inhaled endotoxin and the chemokine, MCP-1. Thus, the biologic response to commonly encountered environmental levels of inhaled LPS is, in part, dependent on functional CD44.
Tak Mak generously provided the CD44-deficient animals used in this study. The authors appreciate the dedication of Jessica Ramsberger and Sandy Hackney with animal husbandry.
This manuscript was supported by grants from the National Institute of Environmental Health Sciences (E12717, ES11961), the National Heart, Lung, and Blood Institute (HL91335, HL67467, HL62472, HL73896), and the National Institute of Allergy and Infectious Diseases (AI058161). This research was supported, in part, by the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences.
Originally Published in Press as DOI: 10.1165/rcmb.2006-0363OC on April 19, 2007
Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.