Surfactant Protein A Is a Principal and Oxidation-sensitiveMicrobial Permeabilizing Factor in the Alveolar Lining Fluid*

Received for publication, October 5, 2004, and in revised form, May 2, 2005Published, JBC Papers in Press, May 12, 2005, DOI 10.1074/jbc.M411344200

Alexander I. Kuzmenko, Huixing Wu, Sijue Wan, and Francis X. McCormack‡

From the Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of CincinnatiSchool of Medicine, Cincinnati, Ohio 45267

We have reported that surfactant protein A kills someGram-negative organisms by increasing membrane per-meability. In this study, we investigated the physiologicimportance of this activity and the effect of oxidativestress on the antimicrobial functions of SP-A in vitroand in vivo. Concentrated bronchoalveolar lavage fluidsfrom SP-A�/� mice increased the permeability of theEscherichia coli K12 cell membrane to a greater extentthan lavage from SP-A�/� animals. Similarly, calcium-dependent surfactant-binding proteins of SP-A�/� miceincreased membrane permeability more than those fromSP-A�/� mice and produced greater zonal killing ofagar-embedded bacteria in a radial diffusion assay. Ex-posure of human SP-A to copper-initiated surfactantphospholipid peroxidation or to free radicals generatedby human neutrophils in vitro increased the level ofSP-A-associated carbonyl moieties and blocked the per-meabilizing function of the protein. We also found thatexposure of mice to 90% O2 for 4 days, sufficient to leadto consumption of glutathione, oxidation of protein thi-ols, and accumulation of airspace protein-associatedcarbonyl moieties, blocked the permeabilizing activityof lavage fluid from SP-A�/� mice. We conclude thatSP-A is a major microbial permeablizing factor in lavagefluid and that oxidative stress inhibits the antibacterialactivity of SP-A by a mechanism that includes oxidativemodification and functional inactivation of the protein.

Surfactant proteins A and D, also known as the pulmonarycollectins, play important roles in the innate immune defenseof the lung, including the agglutination, opsonization, and aug-mentation of intraphagocytic killing of a variety of inhaledpathogens (1). We have recently reported that the pulmonarycollectins also directly inhibit the growth of microorganisms byinduction of membrane permeability (2, 3).

Nosocomial pneumonias are a common complication of pro-longed intensive care unit stays. Supplemental oxygen therapyis extensively used in this setting, resulting in conditions thatfavor formation of reactive oxygen species (ROS)1 and damageto airway cells and extracellular molecules in the alveolar

lining fluid. We recently reported that SP-A is oxidativelymodified and functionally inactivated, with respect to interac-tion with phospholipids, upon exposure of SP-A to lipid peroxi-dation, in vitro (4). We postulate that oxidative damage of SP-Amay also affect the host defense activities of the protein. In thisstudy, we examined the relative physiologic importance of thepermeabilizing activity of SP-A in the lung, and the effects ofoxidizing stimuli on the antimicrobial activity of SP-A and thealveolar lining fluid.

MATERIALS AND METHODS

Reagents—Bovine serum albumin, Chelex 100, cholesterol, cupricsulfate, disodium EDTA, and �-phenantrolinedisulfonic acid were fromSigma. The OxyBlotTM Protein Oxidation Detection Kit was from In-tergen (Purchase, NY). The BCA Protein Assay Reagent Kit was fromPierce (Rockford, IL). 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine, L-phosphatidylcholine from egg yolk, and 1-stearoyl-2-linoleoyl-sn-glyc-ero-3-phosphocholine (18:0–18:2 PC) in chloroform were from AvantiPolar Lipids, Inc. (Alabaster, AL). The alkaline phosphatase substrate,ELF97, was from Molecular Probes, Inc. (Eugene, OR). The thiol-spe-cific probe, ThioGlo1, was from Calbiochem. All other chemicals were ofanalytical grade. Microcon YM-3 MWCO 3,000 Millipore (Bedford, MA)centrifugal filter devices were used for concentration of protein. Spec-tra/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. Kor-fhagen) were developed from embryonic stem cells after disruption ofthe mouse SP-A gene by homologous recombination and maintained bybreeding with Swiss Black mice, as previously reported (5). The SP-Anull allele was bred into the C3H/HeN background through nine gen-erations, as described (2, 3). All comparisons made with the SP-A�/�mice were with age and strain-matched C3H/HEN controls. All animalswere housed in positively ventilated microisolator cages with automaticrecirculating water located in a room with laminar, high efficiencyparticulate-filtered air. The animals received autoclaved food, water,and bedding. Mice were handled in accordance with approved protocolsthrough the Institutional Animal Care and Use Committee at theUniversity of Cincinnati School of Medicine.

SP-A Purification—Human SP-A was isolated from patients withpulmonary alveolar proteinosis, a lung disease associated with theaccumulation of surfactant lipids and proteins. Briefly, SP-A was puri-fied by the method of Suwabe (6) from the cell-free surfactant pellet ofbronchoalveolar lavage by serial sedimentation and resuspension inbuffer containing 5 mM Tris, 150 mM NaCl, and 1 mM Ca2�, release byincubation with 2 mM EDTA, and adsorption of the recalcified super-natant to mannose-Sepharose affinity columns. SP-A was eluted fromthe carbohydrate affinity column using 2 mM EDTA. The purified pro-teins were dialyzed for 2 days against daily changes of 2,000 volumes of5 mM Tris (pH 7.4), 150 mM NaCl and for 1 day against 2,000 volumesof 5 mM Tris (pH 7.4), and stored at �20 °C. The EDTA content of allprotein samples used was measured by the method of Kratochvil, withmodifications as previously reported (4). The average final EDTA con-centration was 5 �M, and was less than 25 �M in all SP-A reagents used.

Membrane Vesicle Preparation—Model surfactant lipids were usedas substrates for lipid oxidation. Unsaturated liposomes (UL) wereprepared by the method of Gregoriadis (7). In brief, an unsaturated lipidmixture (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, cholesterol, eggL-phosphatidylcholine, 18:0–18:2 PC (1:1:0.15:0.15, w/w/w/w)) was dis-

* This work was supported by a Merit Grant from the Department ofVeterans Affairs and National Institutes of Health Grant HL-68861 (toF. X. M.). The costs of publication of this article were defrayed in partby the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

‡ To whom correspondence should be addressed: University of Cin-cinnati, MSB Rm. 6001, 231 Albert Sabin Way, Cincinnati, OH 45267-0564. Tel.: 513-558-4831; Fax: 513-558-4858; E-mail: frank.mccormack@uc.edu.

1 The abbreviations used are: ROS, reactive oxygen species; SP-A,surfactant protein A; UL, unsaturated liposomes; DNPH, dinitrophe-nylhydrazine.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 27, Issue of July 8, pp. 25913–25919, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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solved in CHCl3/methanol (85:15, v/v). The organic solvents were evap-orated to dryness in a rotary evaporator under an N2 atmosphere at20 °C and the lipid film was hydrated in 5 mM Tris, 150 mM NaCl, 3%Chelex-treated buffer (pH 7.5). Multilamellar liposomes were generatedby vigorous vortexing for 5 min.

Assays of Protein Oxidation—Human SP-A was oxidized by coincu-bation with copper-treated UL. Stock solutions of 10 mM CuSO4 werefreshly prepared daily. Reaction mixtures composed of 0.02 mg/ml UL,2 �M CuSO4, and proteins or controls were prepared in 3% Chelex-treated saline (0.9% NaCl) or phosphate-buffered saline for 24 h at20 °C. The mixtures were incubated at 37 °C in a shaking water bathfor 24 h (4). Control reactions that included UL only, or CuSO4 onlywere also performed. Oxidation was monitored by measuring thiobar-bituric acid-reactive substances, using a method adapted from Gelvanand Saltman (8). Samples and malondialdehyde standards were devel-oped by the addition of a solution composed of 0.375% thiobarbituricacid, 15% trichloroacetic acid, and 0.25 N HCl at a volume ratio of 1:2 ofsample/developer. Following incubation at 95 °C for 30 min and centrif-ugation at 16,000 � g for 15 min, an aliquot was read in a spectropho-tometer using a 540-nm filter. An absorption scan (500–570 nm) of boththe malondialdehyde/thiobarbituric acid adducts and the lipid/aldehydethiobarbituric acid adducts indicated that the absorbance at 540 nmwas representative of the peak obtained at the thiobarbituric acidabsorption maximum at 532 nm (not shown).

Analysis of Escherichia coli Cell Wall Permeability—The effect of theSP-A on E. coli cell wall integrity was assessed by determining perme-ability to cleavage activated, fluorescent alkaline phosphatase sub-strate, ELF97. E. coli (A600 nm � �0.5 absorbance units) in 100 �l of 5mM Tris and 150 mM NaCl was incubated with 100 �M ELF97 phos-phatase substrate at 37 °C with 50 �g/ml SP-A from 20 to 90 min. Thechanges in fluorescence intensity were measured at excitation andemission wavelengths of 355 and 535 nm, respectively.

Assays of Protein-associated Carbonyls—Oxidative modification ofSP-A was determined by Western blot analysis (Oxyblot) using anantibody to 2,4-dinitrophenylhydrazine (DNP)-derivatized carbonylgroups. SP-A was incubated with DNP to modify protein-associatedcarbonyls. After size fractionation by 8–16% SDS-polyacrylamide gelelectrophoresis under reducing conditions, protein species were electro-phoretically transferred to nitrocellulose membranes. The membraneswere sequentially incubated with a rabbit anti-DNP IgG and a horse-radish peroxidase-conjugated goat anti-rabbit IgG. Blots were devel-oped by horseradish peroxidase-dependent oxidation of a chemilumi-nescent 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 or96 h. Mice were housed in sealed plastic chamber-cages with free accessto 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 measuredusing a MiniOx-I oxygen analyzer (MSA Medical Products, Pittsburgh,PA). After various intervals of O2 exposure, animals were lavaged andanalyzed for SP-A, GSH, and protein-SH. A small aliquot of lavage wasused to determine total protein content.

Assays of Total Protein SP-A Concentration—Total protein concen-trations 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 IgGagainst rat SP-A using an enzyme-linked immunosorbent assay, aspreviously reported (9). The lower limit of sensitivity of the assay was0.20 ng/ml, and the linear range extended from 0.16 to 10.0 ng/ml.

Reduced Glutathione and Protein Thiol Measurements—Contents ofGSH and protein thiols in the lavage fluid of mice were measured usingthe thiol-specific fluorophore, ThioGlo1 (3). Low molecular weight sub-stances were separated from lavage by filtering through Microcon YM-3MWCO 3,000 Millipore centrifugal filter devices. A 10-�l aliquot oflavage filtrate or lavage in 100 �l of Hepes-buffered saline solution wasincubated with 10 �M ThioGlo1 in the absence or presence of the proteindenaturing agent SDS, respectively. The GSH and thiol-containingproteins were detected using a fluorescent plate reader with excitationand emission wavelengths of 405 and 535 nm, respectively. Thiol-containing proteins were quantified by subtracting fluorescence be-cause of released glutathione (i.e. signal obtained from the lavagefiltrate in the absence of SDS) from total fluorescence (i.e. signal ob-tained 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 at400 � g for 5 min at 4 °C to sediment the cells. Surfactant was sepa-rated from the cell-free supernatant by centrifugation at 45,000 � g for

6 h at 4 °C in the presence of 2 mM CaCl2. Proteins bound to thesurfactant pellet in a calcium-dependent manner were released byincubation with 2 mM EDTA.

Radial Diffusion Assay of E. coli Viability—Inhibition of bacterialgrowth was assessed using a radial diffusion method (10). MoltenSeaKem LE-agarose (0.8–1.0%) (FMC Bioproducts) in 16 ml of buffercontaining 10 mM sodium phosphate and 1.0% LB medium was mixedwith 150 �l of E. coli (A600 0.3 absorbance units) at a temperature of40 °C and allowed to harden by cooling. Agar composed of tryptone (40g/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-�l wells bored in the agar. After overnight incuba-tion at 37 °C, the plates were visually inspected for clearing around thewells.

Preparation of Human Neutrophils—Heparinized blood from healthyvolunteers was separated by discontinuous density-gradient centrifu-gation (11) to obtain neutrophils. The granulocyte-enriched material atthe interface was collected, centrifuged, and washed in Hanks’ balancedsalt solution (pH 7.4). Contaminating erythrocytes were removed byhypotonic lysis, and the washed neutrophils were resuspended in Dul-becco’s modified Eagle’s medium.

Exposure of SP-A to Neutrophil ROS—Neutrophils (2 � 105 cells) inDulbecco’s modified Eagle’s medium were prewarmed to 37 °C for 5 minand luminol (1 mM) was added to a total volume of 0.1 ml. The oxidativeburst was assessed by measurement of chemiluminescence (12) using aVictor II plate reader chemiluminometer (Turku, Finland). SP-A (100�g/ml) was incubated with 2 � 105 neutrophils in 0.1 ml of Dulbecco’smodified Eagle’s medium for 2 h at 37 °C and 5% CO2 and the sampleswere analyzed for oxidative damage by immunoblot-based assessmentof 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 com-parisons between experimental groups. All data are presented asmean � S.E. unless otherwise noted.

RESULTS

Antimicrobial Activity of Calcium-dependent Binding Pro-teins from the Surfactant Pellet of SP-A�/� and SP-A�/�Mice—Experiments were performed to determine the directeffect of alveolar lavage fluids isolated from SP-A�/� andSP-A�/� mice on E. coli growth using a radial diffusionmethod and the results are shown in Fig. 1. SP-A is intimatelyassociated with surfactant phospholipids in the airspace, suchthat less than 1% of protein is found in the unbound state in theaqueous phase. Surfactant pellets were prepared from mice inthe presence of calcium, in a manner that preserves the asso-ciation between SP-A and sedimented phospholipid. Calcium-dependent binding proteins released from the surfactant pelletof SP-A�/� mice (total protein 6.3 �g/well) produced dose-de-pendent zonal clearing; considerably more than the same con-centration of protein eluted from the pellet of SP-A�/� mice.The EDTA containing, protein-free filtrate (molecular weightcutoff of 10,000) from the surfactant pellet of SP-A�/� micehad little effect on E. coli growth. The data indicate that

FIG. 1. Inhibition of E. coli growth by proteins eluted from thesurfactant pellet of SP-A�/� and SP-A�/� mice. Molten agarosewas mixed with E. coli K12, plated in Petri dishes, and allowed to cool.Wells were bored in the agar, proteins (6.5 �g/well) from lavage fluid orprotein-free filtrate were added, and the plates were incubated over-night at 37 °C.

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calcium-dependent surfactant-binding proteins in SP-A�/�mice have greater antibacterial activity than those fromSP-A�/� mice.

Permeabilizing Activity of Lavage and Calcium-dependentSurfactant-binding Proteins—We have previously reportedthat pulmonary collectins induce protein leak in E. coli (2). Atechnique was developed to more rapidly assess cell wall per-meability, using an impermeant, fluorescent substrate (ELF97)for the periplasmic enzyme, alkaline phosphatase. We testedthe physiological role of SP-A in induction of E. coli membranepermeability using this technique and airspace proteins iso-lated from genetically engineered mice. Concentrated lavagefrom SP-A�/� mice increased the permeability of the E. colicell wall in a dose-dependent fashion (Fig. 2). At 1 mg/ml lavageprotein, lavage from SP-A�/� mice induced the permeability ofthe 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 wasalso a dose-dependent increase in the initial rate of membranepermeabilization by lavage proteins from the SP-A-sufficientmouse strains, which was greater than that produced by lavagefrom SP-A�/� mouse strains (Figs. 2B and 3B). To furtherdefine the role of SP-A in permeabilization, we tested theantimicrobial activity of calcium-dependent surfactant-bindingproteins from SP-A�/� and SP-A�/� mice. We found that theEDTA eluate from the surfactant pellet of SP-A�/� mice (0.2mg/ml total protein) permeabilized E. coli to an extent that was3.8-fold greater than untreated E. coli (p � 0.05), comparedwith 1.3-fold (p � 0.05) for the proteins eluted from the surfac-tant pellet from SP-A�/� mice (0.2 mg/ml total protein) (Fig.4A). There was also a significant increase in the initial rate ofmembrane permeabilization by proteins from the surfactantpellet of SP-A�/� versus SP-A�/� mice (Fig. 4B). Taken to-gether, these data are consistent with a role for SP-A in thepermeabilizing activity of alveolar lavage fluid.

Effect of Lipid Peroxidation on Collectin Antimicrobial Activ-ity—At a concentration of 50 �g/ml, human SP-A markedlyincreased intracellular penetration of ELF97, resulting in atime-dependent increase in fluorescence that began at �20 minand increased in a linear fashion (Fig. 5, A and B). SP-A wasthen oxidized using both chemical and cellular free radicalsources and tested for antimicrobial activity. Oxidative damageof human SP-A was first assessed by a Western blot analysistechnique that detects carbonyl adducts (13). Human SP-A hada detectable level of carbonyls at baseline, as previously re-ported (14). Exposure of human SP-A to oxidizing conditionsfurther increased the content of protein-associated carbonyls(Fig. 5C), to a greater extent when the protein was exposed tolipid peroxidation from incubation with 2 �M CuSO4 plus ULthan to CuSO4 alone. There was no effect of UL alone on thecarbonyl content of SP-A.

The ability of human SP-A to increase the permeability of theE. coli cell membrane was blocked by pre-exposure of SP-A tolipid peroxidation (Fig. 5, A and B). There was no effect ofpre-exposure of human SP-A to lipids alone, or CuSO4 alone (at4 �M), on the kinetics (Fig. 5A) or end point (Fig. 5B) of SP-A-induced membrane permeability. The data shown in Fig. 5 alsoindicate that, in the absence of human SP-A, there was noeffect of incubation of the bacteria with lipids alone, CuSO4

alone, or lipids plus CuSO4 on intracellular penetration ofalkaline phosphatase substrate.

The effect of physiological levels of ROS produced by acti-vated granulocytes on SP-A antimicrobial function was nextexamined. Neutrophil-generated ROS oxidize the luminol sub-strate and cause emission of chemiluminescence in a time-de-pendent manner (Fig. 6A). Exposure of SP-A to ROS producedby human neutrophils resulted in an increase in protein-asso-

FIG. 2. Concentration-dependent increases in the permeabili-zation of the E. coli cell wall by lavage fluid isolated from SP-A�/� mice. The kinetics of E. coli cell wall permeability in the pres-ence of various concentrations of proteins from lavage of SP-A�/� mice:0.1 mg/ml (�), 0.2 mg/ml (), 1 mg/ml (E), or no protein (�) are shownin panel A. The initial rate of E. coli cell wall permeability is shown inpanel B. Data are mean � S.E., n � 3. *, p � 0.05. a.u., absorbanceunits.

FIG. 3. Lavage fluid isolated from SP-A�/� mice increases thepermeability of the E. coli cell wall to a greater extent thanlavage of SP-A�/� mice. The kinetics of E. coli cell wall permeabilityin the presence of 1 mg/ml protein from lavage of SP-A�/� mice (�), 1mg/ml protein from lavage of SP-A�/� mice (), or no protein (E) areshown in panel A. The initial rate of E. coli cell wall permeability isshown in panel B. Data are mean � S.E., n � 3. * and # p � 0.05. a.u.,absorbance units.

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ciated carbonyls, consistent with oxidative damage (Fig. 6B).Previous studies have shown that exposure of SP-A to highconcentrations of oxidizing agents results in SP-A fragmenta-tion (15). We found minor amounts (7% by densitometry) ofproteolytic degradation (Fig. 7) in the presence of human neu-trophils. Exposure of SP-A to activated macrophages has beenreported to result in nitration (16). There was no evidence ofnitrotyrosine formation (data not shown) in our neutrophilexposure experiments, however. Pre-exposure of SP-A to neu-trophils decreased the permeabilizing effects of the proteincompared with sham exposed SP-A (Fig. 8A). The initial rate ofE. coli membrane permeability induced by neutrophil-exposedSP-A was considerably less than sham exposed SP-A (Fig. 8B).These results are consistent with a significant effect of oxida-tion of SP-A. We cannot exclude the possibility that the minoramounts of proteolysis seen contributed significantly to the lossof permeabilizing activity.

The Effect of Hyperoxia on the Antimicrobial Activity of Al-veolar Lining Fluid from SP-A�/� Mice—To determinewhether clinically relevant oxidant exposures affect the anti-microbial functions of SP-A in vivo, we next assessed the effectof hyperoxic exposure on the permeabilizing activity of thealveolar lining fluid. SP-A�/� mice were exposed to air or to90% oxygen for 96 h. These hyperoxic conditions were sufficientto cause consumption of glutathione (Fig. 9A, p � 0.01), a3.5-fold decrease in protein thiols (Fig. 9B, p � 0.02), a 7.5-foldincrease in the accumulation of total protein (Fig. 9C, p � 0.01),and marked increase in airway protein-associated carbonylmoieties (Fig. 10). The enzyme-linked immunosorbent assay-quantified concentrations of SP-A in the lavage fluid pre- andpost-oxygen exposure were unchanged (29.5 � 5.2 and 27.6 �2.4 ng/ml, respectively). Exposure of mice to hyperoxia did notresult in nitration of lavage proteins, as assessed by immuno-

precipitation followed by Western blot analysis with nitroty-rosine antibody (data not shown). The permeabilizing activityof lavage fluid isolated from the oxygen-exposed animals wasmarkedly decreased compared with the control (air exposed)mice as shown by kinetic profile (Fig. 11A) and initial rate of

FIG. 4. Proteins eluted from the surfactant pellet of SP-A�/�mice increases the permeability of the E. coli cell wall to agreater extent than the eluate of the surfactant pellet of SP-A�/� mice. The kinetics of E. coli cell wall permeability in the pres-ence of 250 �g/ml protein from the surfactant pellet of SP-A�/� mice(�), 250 �g/ml protein from surfactant pellet of SP-A�/� mice (), or noprotein (E) are shown in panel A. The initial rate of E. coli cell wallpermeability is shown in panel B. Data are mean � S.E., n � 3. * and#, p � 0.05. a.u., absorbance units.

FIG. 5. LPO mediated oxidative damage of human SP-A blocksthe collectin mediated permeability of the E. coli cell wall. SP-Awas preincubated alone, with multilamellar phospholipids, with copper,or with multilamellar phospholipids plus copper for 24 h at 37 °C. Thekinetics of E. coli cell wall permeability upon exposure to: 1) SP-A alone(● ); 2) SP-A preincubated with 20 �g/ml multilamellar phospholipids(); 3) 20 �g/ml multilamellar phospholipids alone (�); 4) 4 �M CuSO4alone (�); 5) SP-A preincubated with 4 �M CuSO4 (�); 6) SP-A prein-cubated with 20 �g/ml multilamellar phospholipids and 4 �M CuSO4(E); 7) 20 �g/ml multilamellar phospholipids and 4 �M CuSO4 alone (�)and in the absence of any addition (�) are shown in panel A. The initialrate of E. coli cell wall permeability is shown in panel B. In panel C,oxidative modification of SP-A that occurred during 24 h exposure ofSP-A at 37 °C as in 1–7 above was determined by Western analysisusing an antibody to DNP-derivatized carbonyl moieties and quantifi-cation by densitometry. Data are mean � S.E., n � 3. * and #, p � 0.05.a.u., absorbance units.

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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 withthe activity of SP-A. We found that the loss of permeabilizingactivity of lavage fluid was not because of serum inhibitors ofpermeabilization because a 1:1 mixture of lavage from oxygen-exposed and sham treated animals was as active as a 1:1mixture of buffer and lavage from sham treated animals(Fig. 12).

DISCUSSION

In this study, we examined the relative physiologic impor-tance of SP-A in the membrane permeabilizing activity of al-

veolar lavage fluid, and the effect of oxidation on SP-A antimi-crobial function. We found that lavage from SP-A�/� mice wasmuch less disruptive to E. coli membranes than lavage from

FIG. 6. ROS produced by human blood neutrophils cause oxi-dative damage of human SP-A. The kinetics of luminol (1 mM)-de-pendent chemiluminescence in the presence of 2 � 105 cells of humanblood neutrophils (E) or in the absence of human blood neutrophils (�)are shown in panel A. The carbonyl content of SP-A was assessedfollowing a 2-h exposure to 5% CO2 alone (lanes 1, 3, and 6) or to SP-Apreincubated with human blood neutrophils (lanes 2, 4, and 7); orhuman blood neutrophils alone (lane 5) was determined by Westernanalysis using an antibody to DNP-derivatized carbonyl moieties. Dataare mean � S.E., n � 3. *, p � 0.05. a.u., absorbance units.

FIG. 7. ROS produced by human blood neutrophils cause mi-nor fragmentation of human SP-A. SP-A was preincubated alone orwith 2 � 105 cells of human blood neutrophils for 2 h at 37 °C, 5% CO2,and size fractionation on an overloaded 8–16% SDS-PAGE gel. Frag-mentation of human SP-A was assessed by Western analysis using anantibody to human SP-A. PMN, polymorphonuclear neutrophils.

FIG. 8. Exposure to human neutrophils blocks SP-A permeabi-lizing activity. SP-A was preincubated alone or with 2 � 105 cells ofhuman blood neutrophils for 2 h at 37 °C, in a 5% CO2 atmosphere. Thekinetics of E. coli cell wall permeability upon exposure to SP-A (�),SP-A preincubated with human blood neutrophils (), and in the ab-sence of any addition (�) are shown in panel A. The initial rate of E. colicell wall permeability is also shown in panel B. Data are mean � S.E.,n � 3. * and #, p � 0.05.

FIG. 9. Exposure of SP-A�/� mice to 90% O2 for 96 h decreasedthe content of reduced glutathione (panel A) and protein thiols(panel B), and increased the total concentration of protein(panel C) in lavage compared with SP-A�/� mice exposed to air.Data are mean � S.E., n � 3. *, p � 0.05.

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SP-A�/� mice, suggesting that SP-A is an important microbialpermeabilizing protein in lavage. The permeabilizing activityof SP-A was dose dependent, and was blocked by oxidativedamage to SP-A, in vivo and in vitro. We conclude that oxida-tive stress inhibits the antibacterial activity of SP-A by a mech-anism that includes oxidative modification and functional in-activation of the protein.

The innate immune defense system of the lung consists of avariety of antimicrobial molecules and phagocytic cells (1),including peptides and proteins that opsonize and permeabilizebacteria (2) and fungal microorganisms (3). Several lines ofevidence suggest that oxidative damage to surfactant compo-

nents have critical effects on surfactant biophysical function,but less is known about the consequences of oxidative stress forcollectin-mediated host defense (4, 18).

We have previously demonstrated that the SP-A exerts po-tent, macrophage-independent antibacterial activity againstrough E. coli strains at physiologically relevant collectin con-centrations, in vitro (2, 3). In this study, the demonstrationthat lavage from SP-A�/� mice has greater permeabilizingand antiproliferative effects than that from SP-A�/� micesuggests an antimicrobial role for the protein, in vivo. Further-more, a significant fraction of the permeabilizing activity oflavage fluid segregated with the surfactant pellet (and SP-A)upon centrifugation, rather than in the aqueous phase, whichcontains the non-phospholipid interacting antimicrobial pep-tides like lysozyme and defensins. The relative physiologicimportance of the direct antimicrobial activities of the collec-tins versus their opsonic and macrophage modifying functionsare important questions that were not addressed in this study.

Our results indicate that oxidative damage of surfactantproteins by lipid peroxidation or ROS generated by inflamma-tory cells in vitro inhibits the permeabilizing activity of SP-A.The changes in protein function were associated with oxidativemodification of SP-A as assessed by carbonyl adduct formation,but not with detectable nitration of the protein. A minoramount of proteolytic degradation occurred upon exposure ofSP-A to human blood neutrophils. A small amount of proteol-ysis can be associated with loss of protein function, especiallyfor oligomeric proteins, and the degree of proteolysis can beunderestimated because small fragments may not interactwith the antibody. Nonetheless, loss of permeabilizing activityupon oxidation exposure is generally consistent with our pre-vious observations regarding the functional consequences of

FIG. 10. Exposure of SP-A�/� mice to 90% O2 for 96 h increasedthe content of lavage protein-associated carbonyls as deter-mined by Western analysis using an antibody to DNP-derivat-ized carbonyl moieties. Each line represents a separate animal.

FIG. 11. The lavage from SP-A�/� mice increased the perme-ability of the E. coli cell wall to a greater extent than lavagefrom SP-A�/� mice exposed to 90% O2. The kinetics of E. coli cellwall permeability in the presence of 1 mg/ml protein from lavage fluidof SP-A�/� mice (�), 1 mg/ml protein from lavage fluid of SP-A�/�mice exposed to 90% O2 for 48 h (), 1 mg/ml protein from lavage fluidof SP-A�/� mice exposed to 90% O2 for 96 h (�), 7.5 mg/ml proteinfrom lavage of SP-A�/� mice exposed to 90% O2 for 96 h (● ) or noadded protein (E) are shown in panel A. The initial rate of E. coli cellwall permeability is shown in panel B. Data are mean � S.E., n � 3.*, p � 0.05.

FIG. 12. Loss of the permeabilizing activity of bronchoalveolarlavage fluid following exposure to hyperoxia is not due to aninhibitor. The kinetics of E. coli cell wall permeability induced by a 1:1mixture of concentrated (1 mg/ml) lavage proteins from SP-A�/� miceexposed to hyperoxia with lavage of sham exposed SP-A�/� mice ()was similar to that caused by a 1:1 mixture of lavage from SP-A�/�mice with buffer (�) (panel A). A no addition protein control is alsoshown (�). The initial rate of E. coli cell wall permeability is shown onpanel B.

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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. Expo-sure of SP-A�/� mice to 90% oxygen for 4 days resulted insignificant decreases in permeabilizing activity of lavage, de-spite a 7.5-fold increase in total protein content. This loss ofantimicrobial activity was not attributed to inhibitors of per-meabilization that may have leaked into the airspace from theserum compartment. We speculate that similar oxygen ten-sions used to treat respiratory failure in patients may inacti-vate antimicrobial proteins such as SP-A in the alveolar liningfluid and render subjects more susceptible to infection. In sum-mary, the data in this study indicate that SP-A is a principalpermeabilizing protein in lavage and that oxidative stress as invitro and in vivo results in protein damage and functionalinactivation of antibacterial properties of SP-A.

Acknowledgments—We thank Tom Korfhagen and Jeffrey Whitsettfor SP-A�/� and SP-A�/� mice.

REFERENCES

1. McCormack, F. X., and Whitsett, J. A. (2002) J. Clin. Investig. 109, 707–7122. 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

3. McCormack, F. X., Gibbons, R., Ward, S. R., Kuzmenko, A., Wu, H., and Deepe,G. S., Jr. (2003) J. Biol. Chem. 278, 36250–36256

4. Kuzmenko, A. I., Wu, H., Bridges, J. P., and McCormack, F. X. (2004) J. LipidRes. 45, 1061–1068

5. 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

6. Suwabe, A., Mason, R. J., and Voelker, D. R. (1991) Am. J. Respir. Cell Mol.Biol. 5, 80–86

7. Gregoriadis, G., and Ryman, B. E. (1972) Biochem. J. 129, 123–1338. Gelvan, D., and Saltman, P. (1990) Biochim. Biophys. Acta 1035, 353–3609. McCormack, F. X., Kuroki, Y., Stewart, J. J., Mason, R. J., and Voelker, D. R.

(1994) J. Biol. Chem. 269, 29801–2980710. Ganz, T., Selsted, M. E., Szklarek, D., Harwig, S. S., Daher, K., Bainton, D. F.,

and Lehrer, R. I. (1985) J. Clin. Investig. 76, 1427–143511. Newman, S. L., Gootee, L., and Gabay, J. E. (1993) J. Clin. Investig. 92,

624–63112. Saeed, F. A., and Castle, G. E. (1998) Clin. Diagn. Lab. Immunol. 5, 740–74313. Levine, R. L., Wehr, N., Williams, J. A., Stadtman, E. R., and Shacter, E.

(2000) Methods Mol. Biol. 99, 15–2414. Wang, G., Bates-Kenney, S. R., Tao, J. Q., Phelps, D. S., and Floros, J. (2004)

Biochemistry 43, 4227–423915. Haddad, I. Y., Ischiropoulos, H., Holm, B. A., Beckman, J. S., Baker, J. R., and

Matalon, S. (1993) Am. J. Physiol. 265, L555–L56416. Zhu, S., Basiouny, K. F., Crow, J. P., and Matalon, S. (2000) Am. J. Physiol.

278, L1025–L103117. Zhu, S., Ware, L. B., Geiser, T., Matthay, M. A., and Matalon, S. (2001) Am. J.

Respir. Crit. Care Med. 163, 166–17218. Mark, L., and Ingenito, E. P. (1999) Am. J. Physiol. 276, L491–L500

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