|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 280, Issue 27, 25913-25919, July 8, 2005
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Surfactant Protein A Is a Principal and Oxidation-sensitive Microbial Permeabilizing Factor in the Alveolar Lining Fluid*
From the Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Cincinnati School of Medicine, Cincinnati, Ohio 45267
Received for publication, October 5, 2004 , and in revised form, May 2, 2005.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
We have reported that surfactant protein A kills some Gram-negative organisms by increasing membrane permeability. In this study, we investigated the physiologic importance of this activity and the effect of oxidative stress on the antimicrobial functions of SP-A in vitro and in vivo. Concentrated bronchoalveolar lavage fluids from SP-A+/+ mice increased the permeability of the Escherichia coli K12 cell membrane to a greater extent than lavage from SP-A-/- animals. Similarly, calcium-dependent surfactant-binding proteins of SP-A+/+ mice increased membrane permeability more than those from SP-A-/- mice and produced greater zonal killing of agar-embedded bacteria in a radial diffusion assay. Exposure of human SP-A to copper-initiated surfactant phospholipid peroxidation or to free radicals generated by human neutrophils in vitro increased the level of SP-A-associated carbonyl moieties and blocked the permeabilizing function of the protein. We also found that exposure of mice to 90% O2 for 4 days, sufficient to lead to consumption of glutathione, oxidation of protein thiols, and accumulation of airspace protein-associated carbonyl moieties, blocked the permeabilizing activity of lavage fluid from SP-A+/+ mice. We conclude that SP-A is a major microbial permeablizing factor in lavage fluid and that oxidative stress inhibits the antibacterial activity of SP-A by a mechanism that includes oxidative modification and functional inactivation of the protein.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Surfactant proteins A and D, also known as the pulmonary collectins, play important roles in the innate immune defense of the lung, including the agglutination, opsonization, and augmentation of intraphagocytic killing of a variety of inhaled pathogens (1). We have recently reported that the pulmonary collectins also directly inhibit the growth of microorganisms by induction of membrane permeability (2, 3).
Nosocomial pneumonias are a common complication of prolonged intensive care unit stays. Supplemental oxygen therapy is extensively used in this setting, resulting in conditions that favor formation of reactive oxygen species (ROS)1 and damage to airway cells and extracellular molecules in the alveolar lining fluid. We recently reported that SP-A is oxidatively modified and functionally inactivated, with respect to interaction with phospholipids, upon exposure of SP-A to lipid peroxidation, in vitro (4). We postulate that oxidative damage of SP-A may also affect the host defense activities of the protein. In this study, we examined the relative physiologic importance of the permeabilizing activity of SP-A in the lung, and the effects of oxidizing stimuli on the antimicrobial activity of SP-A and the alveolar lining fluid.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Reagents—Bovine serum albumin, Chelex 100, cholesterol, cupric sulfate, disodium EDTA, and
-phenantrolinedisulfonic acid were
from Sigma. The OxyBlotTM Protein Oxidation Detection Kit was from
Intergen (Purchase, NY). The BCA Protein Assay Reagent Kit was from Pierce
(Rockford, IL). 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine,
L-phosphatidylcholine from egg yolk, and
1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (18:0–18:2
PC) in chloroform were from Avanti%20Polar%20Lipids">Avanti Polar Lipids, Inc. (Alabaster, AL). The
alkaline phosphatase substrate, ELF97, was from Molecular Probes, Inc.
(Eugene, OR). The thiol-specific probe, ThioGlo1, was from Calbiochem. All
other chemicals were of analytical grade. Microcon YM-3 MWCO 3,000 Millipore
(Bedford, MA) centrifugal filter devices were used for concentration of
protein. Spectra/Por cellulose membranes MWCO 3,500 (Spectrum Laboratories,
Inc., Rancho Dominguez, CA) were used for dialysis. SeaKem LE-agarose was from
FMC Bioproducts (Rockland, ME). Mice—Swiss Black SP-A-/- mice (a gift of J. Whitsett and T. Korfhagen) were developed from embryonic stem cells after disruption of the mouse SP-A gene by homologous recombination and maintained by breeding with Swiss Black mice, as previously reported (5). The SP-A null allele was bred into the C3H/HeN background through nine generations, as described (2, 3). All comparisons made with the SP-A-/- mice were with age and strain-matched C3H/HEN controls. All animals were housed in positively ventilated microisolator cages with automatic recirculating water located in a room with laminar, high efficiency particulate-filtered air. The animals received autoclaved food, water, and bedding. Mice were handled in accordance with approved protocols through the Institutional Animal Care and Use Committee at the University of Cincinnati School of Medicine.
SP-A Purification—Human SP-A was isolated from patients with pulmonary alveolar proteinosis, a lung disease associated with the accumulation of surfactant lipids and proteins. Briefly, SP-A was purified by the method of Suwabe (6) from the cell-free surfactant pellet of bronchoalveolar lavage by serial sedimentation and resuspension in buffer containing 5 mM Tris, 150 mM NaCl, and 1 mM Ca2+, release by incubation with 2 mM EDTA, and adsorption of the recalcified supernatant to mannose-Sepharose affinity columns. SP-A was eluted from the carbohydrate affinity column using 2 mM EDTA. The purified proteins were dialyzed for 2 days against daily changes of 2,000 volumes of 5 mM Tris (pH 7.4), 150 mM NaCl and for 1 day against 2,000 volumes of 5 mM Tris (pH 7.4), and stored at -20 °C. The EDTA content of all protein samples used was measured by the method of Kratochvil, with modifications as previously reported (4). The average final EDTA concentration was 5 µM, and was less than 25 µM in all SP-A reagents used.
Membrane Vesicle Preparation—Model surfactant lipids were used as substrates for lipid oxidation. Unsaturated liposomes (UL) were prepared by the method of Gregoriadis (7). In brief, an unsaturated lipid mixture (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, cholesterol, egg L-phosphatidylcholine, 18:0–18:2 PC (1:1:0.15:0.15, w/w/w/w)) was dissolved in CHCl3/methanol (85:15, v/v). The organic solvents were evaporated to dryness in a rotary evaporator under an N2 atmosphere at 20 °C and the lipid film was hydrated in 5 mM Tris, 150 mM NaCl, 3% Chelex-treated buffer (pH 7.5). Multilamellar liposomes were generated by vigorous vortexing for 5 min.
Assays of Protein Oxidation—Human SP-A was oxidized by coincubation with copper-treated UL. Stock solutions of 10 mM CuSO4 were freshly prepared daily. Reaction mixtures composed of 0.02 mg/ml UL, 2 µM CuSO4, and proteins or controls were prepared in 3% Chelex-treated saline (0.9% NaCl) or phosphate-buffered saline for 24 h at 20 °C. The mixtures were incubated at 37 °C in a shaking water bath for 24 h (4). Control reactions that included UL only, or CuSO4 only were also performed. Oxidation was monitored by measuring thiobarbituric acid-reactive substances, using a method adapted from Gelvan and Saltman (8). Samples and malondialdehyde standards were developed by the addition of a solution composed of 0.375% thiobarbituric acid, 15% trichloroacetic acid, and 0.25 N HCl at a volume ratio of 1:2 of sample/developer. Following incubation at 95 °C for 30 min and centrifugation at 16,000 x g for 15 min, an aliquot was read in a spectrophotometer using a 540-nm filter. An absorption scan (500–570 nm) of both the malondialdehyde/thiobarbituric acid adducts and the lipid/aldehyde thiobarbituric acid adducts indicated that the absorbance at 540 nm was representative of the peak obtained at the thiobarbituric acid absorption maximum at 532 nm (not shown).
Analysis of Escherichia coli Cell Wall Permeability—The
effect of the SP-A on E. coli cell wall integrity was assessed by
determining permeability to cleavage activated, fluorescent alkaline
phosphatase substrate, ELF97. E. coli (A600 nm =
0.5 absorbance units) in 100 µl of 5 mM Tris and 150
mM NaCl was incubated with 100 µM ELF97 phosphatase
substrate at 37 °C with 50 µg/ml SP-A from 20 to 90 min. The changes in
fluorescence intensity were measured at excitation and emission wavelengths of
355 and 535 nm, respectively.
Assays of Protein-associated Carbonyls—Oxidative modification of SP-A was determined by Western blot analysis (Oxyblot) using an antibody to 2,4-dinitrophenylhydrazine (DNP)-derivatized carbonyl groups. SP-A was incubated with DNP to modify protein-associated carbonyls. After size fractionation by 8–16% SDS-polyacrylamide gel electrophoresis under reducing conditions, protein species were electrophoretically transferred to nitrocellulose membranes. The membranes were sequentially incubated with a rabbit anti-DNP IgG and a horseradish peroxidase-conjugated goat anti-rabbit IgG. Blots were developed by horseradish peroxidase-dependent oxidation of a chemiluminescent substrate and visualized using autoradiography.
Hyperoxic Treatment—Adult male and female C3H/HeN mice (n = 15 per group) were exposed either to 90% O2 or to room air for 48 or 96 h. Mice were housed in sealed plastic chamber-cages with free access to food and water. The ambient oxygen tension was maintained at 90% by gas flow through the chamber at 5 liters of O2/min and was measured using a MiniOx-I oxygen analyzer (MSA Medical Products, Pittsburgh, PA). After various intervals of O2 exposure, animals were lavaged and analyzed for SP-A, GSH, and protein-SH. A small aliquot of lavage was used to determine total protein content.
Assays of Total Protein SP-A Concentration—Total protein concentrations were analyzed by the BCA Protein Assay Reagent Kit (Pierce). Absorbance was read at 570 nm against a sample blank.
SP-A levels in lavage were determined with a rabbit polyclonal IgG against rat SP-A using an enzyme-linked immunosorbent assay, as previously reported (9). The lower limit of sensitivity of the assay was 0.20 ng/ml, and the linear range extended from 0.16 to 10.0 ng/ml. Reduced Glutathione and Protein Thiol Measurements—Contents of GSH and protein thiols in the lavage fluid of mice were measured using the thiol-specific fluorophore, ThioGlo1 (3). Low molecular weight substances were separated from lavage by filtering through Microcon YM-3 MWCO 3,000 Millipore centrifugal filter devices. A 10-µl aliquot of lavage filtrate or lavage in 100 µl of Hepes-buffered saline solution was incubated with 10 µM ThioGlo1 in the absence or presence of the protein denaturing agent SDS, respectively. The GSH and thiol-containing proteins were detected using a fluorescent plate reader with excitation and emission wavelengths of 405 and 535 nm, respectively. Thiol-containing proteins were quantified by subtracting fluorescence because of released glutathione (i.e. signal obtained from the lavage filtrate in the absence of SDS) from total fluorescence (i.e. signal obtained from lavage in the presence of SDS).
Assays of Surfactant Pellet Preparation—After tracheal cannulation, lungs were lavaged 3 times with 1 ml of sterile 5 mM Tris and 150 mM NaCl. Lavages from four to five mice were pooled and centrifuged at 400 x g for 5 min at 4 °C to sediment the cells. Surfactant was separated from the cell-free supernatant by centrifugation at 45,000 x g for 6 h at 4 °C in the presence of 2 mM CaCl2. Proteins bound to the surfactant pellet in a calcium-dependent manner were released by incubation with 2 mM EDTA.
|
Radial Diffusion Assay of E. coli Viability—Inhibition of bacterial growth was assessed using a radial diffusion method (10). Molten SeaKem LE-agarose (0.8–1.0%) (FMC Bioproducts) in 16 ml of buffer containing 10 mM sodium phosphate and 1.0% LB medium was mixed with 150 µl of E. coli (A600 0.3 absorbance units) at a temperature of 40 °C and allowed to harden by cooling. Agar composed of tryptone (40 g/liter), yeast extract (20 g/liter), and agarose (1%) was layered on top. Albumin, lysozyme (0.1 and 1.0 mg/ml), or concentrated lavage (1 mg/ml) were added to 5-µl wells bored in the agar. After overnight incubation at 37 °C, the plates were visually inspected for clearing around the wells.
Preparation of Human Neutrophils—Heparinized blood from healthy volunteers was separated by discontinuous density-gradient centrifugation (11) to obtain neutrophils. The granulocyte-enriched material at the interface was collected, centrifuged, and washed in Hanks' balanced salt solution (pH 7.4). Contaminating erythrocytes were removed by hypotonic lysis, and the washed neutrophils were resuspended in Dulbecco's modified Eagle's medium.
Exposure of SP-A to Neutrophil ROS—Neutrophils (2 x 105 cells) in Dulbecco's modified Eagle's medium were prewarmed to 37 °C for 5 min and luminol (1 mM) was added to a total volume of 0.1 ml. The oxidative burst was assessed by measurement of chemiluminescence (12) using a Victor II plate reader chemiluminometer (Turku, Finland). SP-A (100 µg/ml) was incubated with 2 x 105 neutrophils in 0.1 ml of Dulbecco's modified Eagle's medium for 2 h at 37 °C and 5% CO2 and the samples were analyzed for oxidative damage by immunoblot-based assessment of protein-associated carbonyls, proteolytic degradation using an anti-human SP-A antibody (9), and nitration using an nitrotyrosine antibody (Cayman Chemical).
Statistical Analysis—The analysis of variance test was used for comparisons between experimental groups. All data are presented as mean ± S.E. unless otherwise noted.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Antimicrobial Activity of Calcium-dependent Binding Proteins from the Surfactant Pellet of SP-A+/+ and SP-A-/- Mice—Experiments were performed to determine the direct effect of alveolar lavage fluids isolated from SP-A+/+ and SP-A-/- mice on E. coli growth using a radial diffusion method and the results are shown in Fig. 1. SP-A is intimately associated with surfactant phospholipids in the airspace, such that less than 1% of protein is found in the unbound state in the aqueous phase. Surfactant pellets were prepared from mice in the presence of calcium, in a manner that preserves the association between SP-A and sedimented phospholipid. Calcium-dependent binding proteins released from the surfactant pellet of SP-A+/+ mice (total protein 6.3 µg/well) produced dose-dependent zonal clearing; considerably more than the same concentration of protein eluted from the pellet of SP-A-/- mice. The EDTA containing, protein-free filtrate (molecular weight cutoff of 10,000) from the surfactant pellet of SP-A+/+ mice had little effect on E. coli growth. The data indicate that calcium-dependent surfactant-binding proteins in SP-A+/+ mice have greater antibacterial activity than those from SP-A-/- mice.
|
), 0.2
mg/ml (
), 1 mg/ml (
), or no protein (
) are shown in
panel A. The initial rate of E. coli cell wall permeability
is shown in panel B. Data are mean ± S.E., n = 3.
*, p < 0.05. a.u., absorbance units.Permeabilizing Activity of Lavage and Calcium-dependent Surfactant-binding Proteins—We have previously reported that pulmonary collectins induce protein leak in E. coli (2). A technique was developed to more rapidly assess cell wall permeability, using an impermeant, fluorescent substrate (ELF97) for the periplasmic enzyme, alkaline phosphatase. We tested the physiological role of SP-A in induction of E. coli membrane permeability using this technique and airspace proteins isolated from genetically engineered mice. Concentrated lavage from SP-A+/+ mice increased the permeability of the E. coli cell wall in a dose-dependent fashion (Fig. 2). At 1 mg/ml lavage protein, lavage from SP-A+/+ mice induced the permeability of the E. coli cell wall 14.8-fold (p < 0.001) compared with 3.6-fold (p > 0.05) for lavage from SP-A-/- mice (Fig. 3A). There was also a dose-dependent increase in the initial rate of membrane permeabilization by lavage proteins from the SP-A-sufficient mouse strains, which was greater than that produced by lavage from SP-A-/- mouse strains (Figs. 2B and 3B). To further define the role of SP-A in permeabilization, we tested the antimicrobial activity of calcium-dependent surfactant-binding proteins from SP-A+/+ and SP-A-/- mice. We found that the EDTA eluate from the surfactant pellet of SP-A+/+ mice (0.2 mg/ml total protein) permeabilized E. coli to an extent that was 3.8-fold greater than untreated E. coli (p < 0.05), compared with 1.3-fold (p > 0.05) for the proteins eluted from the surfactant pellet from SP-A-/- mice (0.2 mg/ml total protein) (Fig. 4A). There was also a significant increase in the initial rate of membrane permeabilization by proteins from the surfactant pellet of SP-A+/+ versus SP-A-/- mice (Fig. 4B). Taken together, these data are consistent with a role for SP-A in the permeabilizing activity of alveolar lavage fluid.
|
Effect of Lipid Peroxidation on Collectin Antimicrobial Activity—At a concentration of 50 µg/ml, human SP-A markedly increased intracellular penetration of ELF97, resulting in a time-dependent increase in fluorescence that began at
20 min and increased in a linear
fashion (Fig. 5, A and
B). SP-A was then oxidized using both chemical and
cellular free radical sources and tested for antimicrobial activity. Oxidative
damage of human SP-A was first assessed by a Western blot analysis technique
that detects carbonyl adducts
(13). Human SP-A had a
detectable level of carbonyls at baseline, as previously reported
(14). Exposure of human SP-A
to oxidizing conditions further increased the content of protein-associated
carbonyls (Fig. 5C),
to a greater extent when the protein was exposed to lipid peroxidation from
incubation with 2 µM CuSO4 plus UL than to
CuSO4 alone. There was no effect of UL alone on the carbonyl
content of SP-A. The ability of human SP-A to increase the permeability of the E. coli cell membrane was blocked by pre-exposure of SP-A to lipid peroxidation (Fig. 5, A and B). There was no effect of pre-exposure of human SP-A to lipids alone, or CuSO4 alone (at 4 µM), on the kinetics (Fig. 5A) or end point (Fig. 5B) of SP-A-induced membrane permeability. The data shown in Fig. 5 also indicate that, in the absence of human SP-A, there was no effect of incubation of the bacteria with lipids alone, CuSO4 alone, or lipids plus CuSO4 on intracellular penetration of alkaline phosphatase substrate.
The effect of physiological levels of ROS produced by activated
granulocytes on SP-A antimicrobial function was next examined.
Neutrophil-generated ROS oxidize the luminol substrate and cause emission of
chemiluminescence in a time-dependent manner
(Fig. 6A). Exposure of
SP-A to ROS produced by human neutrophils resulted in an increase in
protein-associated carbonyls, consistent with oxidative damage
(Fig. 6B). Previous
studies have shown that exposure of SP-A to high concentrations of oxidizing
agents results in SP-A fragmentation
(15). We found minor amounts
(
7% by densitometry) of proteolytic degradation
(Fig. 7) in the presence of
human neutrophils. Exposure of SP-A to activated macrophages has been reported
to result in nitration (16).
There was no evidence of nitrotyrosine formation (data not shown) in our
neutrophil exposure experiments, however. Pre-exposure of SP-A to neutrophils
decreased the permeabilizing effects of the protein compared with sham exposed
SP-A (Fig. 8A). The
initial rate of E. coli membrane permeability induced by
neutrophil-exposed SP-A was considerably less than sham exposed SP-A
(Fig. 8B). These
results are consistent with a significant effect of oxidation of SP-A. We
cannot exclude the possibility that the minor amounts of proteolysis seen
contributed significantly to the loss of permeabilizing activity.
|
The Effect of Hyperoxia on the Antimicrobial Activity of Alveolar Lining Fluid from SP-A+/+ Mice—To determine whether clinically relevant oxidant exposures affect the antimicrobial functions of SP-A in vivo, we next assessed the effect of hyperoxic exposure on the permeabilizing activity of the alveolar lining fluid. SP-A+/+ mice were exposed to air or to 90% oxygen for 96 h. These hyperoxic conditions were sufficient to cause consumption of glutathione (Fig. 9A, p < 0.01), a 3.5-fold decrease in protein thiols (Fig. 9B, p < 0.02), a 7.5-fold increase in the accumulation of total protein (Fig. 9C, p < 0.01), and marked increase in airway protein-associated carbonyl moieties (Fig. 10). The enzyme-linked immunosorbent assay-quantified concentrations of SP-A in the lavage fluid pre- and post-oxygen exposure were unchanged (29.5 ± 5.2 and 27.6 ± 2.4 ng/ml, respectively). Exposure of mice to hyperoxia did not result in nitration of lavage proteins, as assessed by immunoprecipitation followed by Western blot analysis with nitrotyrosine antibody (data not shown). The permeabilizing activity of lavage fluid isolated from the oxygen-exposed animals was markedly decreased compared with the control (air exposed) mice as shown by kinetic profile (Fig. 11A) and initial rate of E. coli cell wall permeabilization (Fig. 11B).
|
|
|
Exposure of mice to hyperoxia resulted in significant air space accumulation of serum proteins such as albumin (17). Under these conditions, protein inhibitors might interfere with the activity of SP-A. We found that the loss of permeabilizing activity of lavage fluid was not because of serum inhibitors of permeabilization because a 1:1 mixture of lavage from oxygen-exposed and sham treated animals was as active as a 1:1 mixture of buffer and lavage from sham treated animals (Fig. 12).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In this study, we examined the relative physiologic importance of SP-A in the membrane permeabilizing activity of alveolar lavage fluid, and the effect of oxidation on SP-A antimicrobial function. We found that lavage from SP-A-/- mice was much less disruptive to E. coli membranes than lavage from SP-A+/+ mice, suggesting that SP-A is an important microbial permeabilizing protein in lavage. The permeabilizing activity of SP-A was dose dependent, and was blocked by oxidative damage to SP-A, in vivo and in vitro. We conclude that oxidative stress inhibits the antibacterial activity of SP-A by a mechanism that includes oxidative modification and functional inactivation of the protein.
|
|
|
|
The innate immune defense system of the lung consists of a variety of antimicrobial molecules and phagocytic cells (1), including peptides and proteins that opsonize and permeabilize bacteria (2) and fungal microorganisms (3). Several lines of evidence suggest that oxidative damage to surfactant components have critical effects on surfactant biophysical function, but less is known about the consequences of oxidative stress for collectin-mediated host defense (4, 18).
|
We have previously demonstrated that the SP-A exerts potent, macrophage-independent antibacterial activity against rough E. coli strains at physiologically relevant collectin concentrations, in vitro (2, 3). In this study, the demonstration that lavage from SP-A+/+ mice has greater permeabilizing and antiproliferative effects than that from SP-A-/- mice suggests an antimicrobial role for the protein, in vivo. Furthermore, a significant fraction of the permeabilizing activity of lavage fluid segregated with the surfactant pellet (and SP-A) upon centrifugation, rather than in the aqueous phase, which contains the non-phospholipid interacting antimicrobial peptides like lysozyme and defensins. The relative physiologic importance of the direct antimicrobial activities of the collectins versus their opsonic and macrophage modifying functions are important questions that were not addressed in this study.
Our results indicate that oxidative damage of surfactant proteins by lipid peroxidation or ROS generated by inflammatory cells in vitro inhibits the permeabilizing activity of SP-A. The changes in protein function were associated with oxidative modification of SP-A as assessed by carbonyl adduct formation, but not with detectable nitration of the protein. A minor amount of proteolytic degradation occurred upon exposure of SP-A to human blood neutrophils. A small amount of proteolysis can be associated with loss of protein function, especially for oligomeric proteins, and the degree of proteolysis can be underestimated because small fragments may not interact with the antibody. Nonetheless, loss of permeabilizing activity upon oxidation exposure is generally consistent with our previous observations regarding the functional consequences of SP-A oxidative modification on lipid protein interactions (4).
Next we studied the in vivo effect of hyperoxic exposure on antibacterial properties of lavage from SP-A+/+ mice. Exposure of SP-A+/+ mice to 90% oxygen for 4 days resulted in significant decreases in permeabilizing activity of lavage, despite a 7.5-fold increase in total protein content. This loss of antimicrobial activity was not attributed to inhibitors of permeabilization that may have leaked into the airspace from the serum compartment. We speculate that similar oxygen tensions used to treat respiratory failure in patients may inactivate antimicrobial proteins such as SP-A in the alveolar lining fluid and render subjects more susceptible to infection. In summary, the data in this study indicate that SP-A is a principal permeabilizing protein in lavage and that oxidative stress as in vitro and in vivo results in protein damage and functional inactivation of antibacterial properties of SP-A.
|
FOOTNOTES |
|---|
* This work was supported by a Merit Grant from the Department of Veterans Affairs and National Institutes of Health Grant HL-68861 (to F. X. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: University of Cincinnati, MSB Rm.
6001, 231 Albert Sabin Way, Cincinnati, OH 45267-0564. Tel.: 513-558-4831;
Fax: 513-558-4858; E-mail:
frank.mccormack{at}uc.edu.
1 The abbreviations used are: ROS, reactive oxygen species; SP-A, surfactant
protein A; UL, unsaturated liposomes; DNPH, dinitrophenylhydrazine.
|
ACKNOWLEDGMENTS |
|---|
We thank Tom Korfhagen and Jeffrey Whitsett for SP-A+/+ and SP-A-/- mice.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
- McCormack, F. X., and Whitsett, J. A. (2002) J. Clin. Investig. 109,707 -712[CrossRef][Medline] [Order article via Infotrieve]
- Wu, H., Kuzmenko, A., Wan, S., Schaffer, L., Weiss, A., Fisher, J. H., Kim, K. S., and McCormack, F. X. (2003) J. Clin. Investig. 111,1589 -1602[CrossRef][Medline] [Order article via Infotrieve]
- McCormack, F. X., Gibbons, R., Ward, S. R., Kuzmenko, A., Wu, H.,
and Deepe, G. S., Jr. (2003) J. Biol.
Chem. 278,36250
-36256
[Abstract/Free Full Text] - Kuzmenko, A. I., Wu, H., Bridges, J. P., and McCormack, F. X.
(2004) J. Lipid Res.
45,1061
-1068
[Abstract/Free Full Text] - Korfhagen, T. R., Bruno, M. D., Ross, G. F., Huelsman, K. M.,
Ikegami, M., Jobe, A. H., Wert, S. E., Stripp, B. R., Morris, R. E., Glasser,
S. W., Bachurski, C. J., Iwamoto, H. S., and Whitsett, J. A.
(1996) Proc. Natl. Acad. Sci. U. S. A.
93,9594
-9599
[Abstract/Free Full Text] - Suwabe, A., Mason, R. J., and Voelker, D. R. (1991) Am. J. Respir. Cell Mol. Biol. 5, 80-86
- Gregoriadis, G., and Ryman, B. E. (1972) Biochem. J. 129,123 -133[Medline] [Order article via Infotrieve]
- Gelvan, D., and Saltman, P. (1990) Biochim. Biophys. Acta 1035,353 -360[Medline] [Order article via Infotrieve]
- McCormack, F. X., Kuroki, Y., Stewart, J. J., Mason, R. J., and
Voelker, D. R. (1994) J. Biol. Chem.
269,29801
-29807
[Abstract/Free Full Text] - Ganz, T., Selsted, M. E., Szklarek, D., Harwig, S. S., Daher, K., Bainton, D. F., and Lehrer, R. I. (1985) J. Clin. Investig. 76,1427 -1435
- Newman, S. L., Gootee, L., and Gabay, J. E. (1993) J. Clin. Investig. 92,624 -631
- Saeed, F. A., and Castle, G. E. (1998)
Clin. Diagn. Lab. Immunol.
5, 740-743
[Abstract/Free Full Text] - Levine, R. L., Wehr, N., Williams, J. A., Stadtman, E. R., and Shacter, E. (2000) Methods Mol. Biol. 99, 15-24[Medline] [Order article via Infotrieve]
- Wang, G., Bates-Kenney, S. R., Tao, J. Q., Phelps, D. S., and Floros, J. (2004) Biochemistry 43,4227 -4239[CrossRef][Medline] [Order article via Infotrieve]
- Haddad, I. Y., Ischiropoulos, H., Holm, B. A., Beckman, J. S., Baker, J. R., and Matalon, S. (1993) Am. J. Physiol. 265,L555 -L564
- Zhu, S., Basiouny, K. F., Crow, J. P., and Matalon, S. (2000) Am. J. Physiol. 278,L1025 -L1031
- Zhu, S., Ware, L. B., Geiser, T., Matthay, M. A., and Matalon, S.
(2001) Am. J. Respir. Crit. Care Med.
163,166
-172
[Abstract/Free Full Text] - Mark, L., and Ingenito, E. P. (1999) Am. J. Physiol. 276,L491 -L500
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  T. M. Umstead, W. M. Freeman, V. M. Chinchilli, and D. S. Phelps Age-related changes in the expression and oxidation of bronchoalveolar lavage proteins in the rat Am J Physiol Lung Cell Mol Physiol, January 1, 2009; 296(1): L14 - L29. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. S. George, K. L. Goss, D. K. Meyerholz, F. S. Lamb, and J. M. Snyder Surfactant-Associated Protein A Provides Critical Immunoprotection in Neonatal Mice Infect. Immun., January 1, 2008; 76(1): 380 - 390. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Starosta, E. Rietschel, K. Paul, U. Baumann, and M. Griese Oxidative Changes of Bronchoalveolar Proteins in Cystic Fibrosis. Chest, February 1, 2006; 129(2): 431 - 437. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |