Lung Surfactant Suppresses Oxygen-Dependent ... - uwa biophysics

0022-1767/93/1506-2391$02.00/0 The Journal oi Immunology Copyright 0 1993 by The American Association of Immunologists

Vol 150, 2391-2400, No. 6, March 15, 1993 Printed in U.S.A.

Lung Surfactant Suppresses Oxygen-Dependent Bactericidal Functions of Human Blood Monocytes by Inhibiting the Assembly of the NADPH Oxidase’ Minke F. Geertsma, Hillie R. Broos, Maria T. van den Barselaar, Peter Ralph van Furth2

H. Nibbering, and

Department of Infectious Diseases, University Hospital, Leiden, The Netherlands

ABSTRACT. Surfactant is known to lower the surface tension in alveoliand affects the antibacterial functionsof alveolar and peritoneal macrophages. We investigated the effects of surfactant on the bactericidal functions and oxidative metabolism of human bloodmonocytes andgranulocytes. Monocytes incubatedwith surfactant ingest this material and subsequently exhibit an impaired ability to kill ingested bacteria. Granulocytes incubated with surfactant do not ingest this material, and their bactericidal functionsare not affected. However, granulocytes that have ingested surfactant-coated Staphylococcus aureus display an impaired ability tokill these bacteria. Moreover, in monocytes and granulocytes that contain surfactant-the

latter by ingestion of surfactant-coated S. a u r e u r t h e intracellular

production of H,Oz is impaired due to inhibition of the assembly of the NADPH oxidase. Together these results demonstrate that surfactant inside monocytes and granulocytes inhibits the capacity of these cells to kill bacteria intracellularly by impairing oxygen-dependent killing mechanisms. journal of Immunology, 1993, 150: 2391.

U

nder steady state conditions, alveolar macrophages are the predominant phagocytes of the lower airways and the alveoli. These cells function as scavengers and release proteases, reactive oxygenintermediates, arachidonic acidmetabolites, chemotactic factors, and cytokines (1). Alveolar macrophages reside in a microenvironment rich in surfactant, the main function of which is to lower surface tension in the alveoli (2). In a previous study, we showed that the capacity of mouse alveolar macrophages to kill various bacteria intracellularly is very low compared tothat of peritoneal macrophages and that the capacityof peritoneal macrophages tokill ingested bacteria is severely impaired after incubation with surfac-

tant (3). Others have also reported that surfactant affects the antibacterial functions of alveolar macrophages (4-7). Human alveolar macrophages exhibit a limited bactericidal activity compared to monocytes and granulocytes (8). Under normal conditions, monocytes migrate to the lungs and enter the alveoli where they differentiate into alveolar macrophages (9, 10).At that site, the monocytes come into contactwithsurfactant.Alveolar macrophages contain abundant amounts ofthis material (1 1). Duringan inflammatory reaction in the lungs due to an infection, increased numbers of monocytes as well as granulocytescan enterthe alveoli (9, 12, 13). The present study focused on the question of whether surfactant affects the bactericidal functions of these phagocytes and if so, which mechanisms are involved.

Receivedfor publication September 11, 1992. Accepted for publication December 17, 1992. The costs oi publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advenisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ This study was

supported by the Netherlands Asthma Foundation.

’Address correspondence and reprint requests to

R. vanFurth, University Hospital, Department oi Infectious Diseases, Building 1, C5-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands.

Materials and Methods Bacteria

Staphylococcus aureus (type 42D), S. epidermidis (serotype 4), Escherichia coli (type 054), Streptococcus pyogenes (group A), and Streptococcus pneumoniue (serotype

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SURFACTANT SUPPRESSES MONOCYTE BACTERICIDAL FUNCTIONS

23) were cultured overnightin Nutrient broth no. 2. (Oxoid Ltd., Basingstoke, United Kingdom) at 37°C. Bacteriawere harvested by centrifugation at 1500X g, washed twice with PBS, and resuspended in HBSS containing 0.1% gelatin (gelatin-HBSS) to a concentration of about 1 X lo7 microorganisms/ml.

lipid in surfactant; since surfactant consists of almost 90% phospholipids (16, 17), the concentration of organic phosphate is representative of the concentration of surfactant. Humansurfactant was prepared from BAL fluidasdescribed for sheep surfactant. Labeling of surfactant with FITC

Cells

Buffy coats from blood of healthy donors were diluted in PBS and subjected to differential centrifugation at 400 X g for 20 min on a Ficoll-Hypaque gradient( p = 1.077 g/ml; Pharmacia, Uppsala, Sweden) (14). The monocytelymphocyte layer was washed four times with PBS containing 0.5 U heparin/ml and suspended to a concentration of 1 X lo7 monocytes/ml gelatin-HBSS. This preparation consisted of about 30% monocytes,65% lymphocytes, and less than5% granulocytes. After resuspensionof the FicollHypaque pellet in PBS, the granulocytes were purified by plasma-sterile(Fresenius AG, Bad Hamberg,Germany) sedimentation for 10 min at 37°C followed by removal of the E by hypotonic lysis with distilled water; a cell suspension of 1 X lo7 granulocytes/ml gelatin-HBSS was then prepared. Viability of monocytes and granulocytes, as determined by trypan blue exclusion, was more than 95%. Sheep monocytes were isolated from fresh sheep blood as described above for human monocytes, except that differential centrifugationof blood onthe Ficoll-Hypaque gradient was performed at 650 X g for 45 min. Human alveolar macrophages were isolated from BAL3 fluid from lung segments with no underlying pathologic condition that were collected after informed consent from patients who underwent BAL for diagnostic reasons. The final BAL cell suspension consisted of about 90 to 95% alveolar macrophages, 4 to 9% lymphocytes, and 1% granulocytes. Viability of the cells exceeded 95%, as measured by trypan blue exclusion. Isolation of surfactant

Surfactant was obtained from freshly excised sheep lungs by repeated lavage with1.5 to 2 litersof saline. The airways were filled with saline until fully inflated,and then the fluid was removed. The pooled lavage fluid was centrifuged at 750 X g for 20 min to remove cells and debris, the supernatant was collected and centrifuged at 21,000 X g for 2 h, and the resulting pellet was resuspended in a small volume of saline and stored at -20°C. The concentration of surfactant was expressed in mM organic phosphate, which was determined by Dr. J. Egberts (Department of Obstetrics, University Hospital, Leiden,The Netherlands) according to the method of Bartlett (15). The amount of organic phosphate is proportional to the total amount of phospho-

’

Abbrevlations used in this paper: BAL, bronchoalveolar lavage; DCFH-DA, 2’,7’-dichlorofluorescin diacetate; DPI, diphenyleneiodonium.

FITC-labeled surfactant (FITC-surfactant)was prepared by dialysis of surfactantataconcentration of15 pmol phospholipid/ml 0.05 M carbonate buffer (pH 9.5) against 0.1 mg FITC (Isomer I; Sigma)/mlO.O5 M carbonate buffer for 18 h at 4°C. Free FITC was removed by centrifugation of the suspension at 21,000 X g for 2 h; the material in the pellet was then dialyzed four times against 0.05 M carbonate buffer. The finalsuspension was centrifugedat 21,000 X g for 2 h andthe FITC-surfactant was suspended in saline at a concentration of about 30 mM. Incubation of monocytes and granulocytes with surfactant

Monocytes or granulocytes at a concentration of 1 X 107/ml gelatin-HBSS were incubated with various concentrations of surfactantfor 30 min at 37°Cunderslow rotation (4 rpm). After three washes with gelatin-HBSS and centrifugation at250 X g to remove excess surfactant, the cells were resuspended to a concentrationof 1 X 107/ml gelatinHBSS. Viability of surfactant-incubatedmonocytesand granulocytes exceeded 90%, as measured by trypan blue exclusion. Control monocytes and granulocytes were incubated with saline, the diluent of surfactant, under identical conditions. Measurement of the uptake of FITC-surfactant by monocytes and granulocytes

After incubationof monocytes or granulocytes with various concentrations of FITC-surfactant for various time periods at 37”C, the cells were washed four times with PBS, fixed with 1% paraformaldehyde in PBS supplemented with 1% BSA (PBS-BSA), and resuspended in PBS-BSA after one wash to remove paraformaldehyde. The amount of FITCsurfactant associatedwith monocytes and granulocytes was measured with a FACStar (Becton-Dickinson,Mountain View, CA) equippedwith an argon-ion laser (excitation wavelength at 488 nm, laser power 300 mW) and a band pass filter of 530 nm (width, 20 nm). In each sample, 1 X lo4 cells were analyzed; the results are expressed as the mean of their fluorescence intensity. The localization of FITC-surfactant in phagocytes was studied by means of confocal laser scanning fluorescence microscopy. The plasma membranes of monocytes were stained immunocytochemically with the mAb 63D3 that recognizesthe CD14 Ag (IS), biotinylatedhorse-antimouse IgG, and streptavidin conjugated with phycoerythrin and Texas red (Southern Biotechnology Associates, Inc.,

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journal of Immunology

Birmingham, AL). To acquire dual wavelength images, an excitation wavelength of 488 nm, a short-pass 560-nm filter, and a long-pass 630-nm filter were used in the confocal laser scanning fluorescence microscope (modelMRC-500; Bio-Rad Laboratories, Richmond,CA). Theresults are presented as photographs of computer images. Serum

Serum was prepared from the blood of healthy donors with blood group AB. The blood was clotted for 1 h at room temperature and centrifuged for20 min at 1500 X g; serum was then collected and stored in 0.5 ml-aliquots at -70°C. Phagocytosis of bacteria

The assayforphagocytosiswasperformedasdescribed elsewhere ( 1 9). In short, 200 p1 of the bacteria suspension containing I X IO7 bacterialml, 200 y1 of the monocyte or granulocyte suspension containing 1 X lo7cells/ml, and 40 pl fresh serum were incubated at 37°C under slow rotation (4 rpm). At the indicatedtimes,a 100 pl-sample of the mixture was transferred to avial containing 900 pl ice-cold gelatin-HBSS tostop phagocytosis. After centrifugationfor 4 min at 110 X g to separateextracellularandcellassociated bacteria, the number of viable bacteria in the supernatant was determinedby a microbiologic plate method. Phagocytosis is expressedas the percentage decreasein the number of viable extracellular bacteria. To demonstrate that bacteria are truly ingested by and not merely adherent to the cells, monocytes preincubated with surfactant were allowedto phagocytose S. aureus for 3min. The mixture was then washed three times with ice-cold gelatin-HBSS to remove noningested bacteria, and the cells with bacteria were exposed to 1 U lysostaphidml (Sigma) for 5 min at 4°C to lyse adherent S.aureus; cytocentrifuge preparations were then made. Next, the number of ingested bacteria per 100cells before and after lysostaphin treatment was assessed by microscopy, andthenumber of celladherent s. aureus was calculated (20). Pre-opsonization of bacteria

Bacteria were pre-opsonized with10% serum for 30 min at 37°C under rotation (4 rpm), excess serumwas removed by centrifugation, andthe bacteria were resuspendedin HBSSgel to a concentration of 1 X 107/ml. Where indicated, 1 X lo7 pre-opsonized S. aureuslml were incubated with 4 mM surfactant for 30 min at 37"C, and then excess surfactant was removedby centrifugation; these bacteria were called pre-opsonized surfactant-coated bacteria. The coating of bacteria with surfactant was confirmed using FITCsurfactant and fluorescence microscopy. Intracellular killing of bacteria

The assay for intracellular killing was performed, as described previously (19). In short, 200 p1 pre-opsonized bac-

teria and 200 yl monocytes or granulocytes (1 X 107/ml) were incubatedfor3min at 37°Cunderslowrotation; phagocytosis was stopped by shaking the tube in crushed ice. The noningested bacteria were removedby differential centrifugation and washing. Then 5 X lo6 phagocytes/ml containing ingested bacteria were reincubated in the presence of 10%AB serum at 37°C under rotation (4 rpm). At the indicated intervals, a 100 pl-sample was transferred to a vial containing 900 pl ice-cold water with 0.01% BSA, and the leukocytes were disrupted by vigorous shaking on a vortex mixer for 1 min. After serial dilution, the numbers of viable intracellular bacteria were determined microbiologically. At the end of the experiment, 1800pl HzO-BSA were added to the remaining cell suspension (200 pl), the cells were disrupted, and the number of intracellular viable bacteria determined as described above. Intracellular killing is expressed as the percentage decrease in the number of viable intracellular bacteria. Incubation of monocytes with diphenyleneiodonium

To suppressoxygen-dependentkillingmechanisms, 1 X lo7 monocytes/ml were incubated with 5 yM NADPH oxidase inhibitor DPI (21), a generous gift from Dr. A. R. Cross (Department of Biochemistry, University of Bristol, Bristol, United Kingdom), for 15 min at 37°C.As a control, monocytes were incubated with 0.5% DMSO, the diluent of DPI. Measurement of extracellular and intracellular H 2 0 2 production

H202 release by phagocytes without a stimulus and upon stimulation with 1,5, or25 ng PMA/ml was assayed by the horseradishperoxidase-mediatedHZO2-dependentoxidation of homovanillic acid (22); the results are expressed as nmol H2O2/(IO6 cells X 60 min). The H202-specificprobe DCFH-DA (23) was used to measure the intracellular production of H2OZupon stimulation with 100 ng PMAlml. Briefly, after incubation of monocytes or granulocytes at a concentration of 1 X 106/ml with 5 pM DCFH-DA (Eastman Kodak, Rochester, NY) for 15 min at 37"C, PMA was added to the cell suspension. The amount of the H202 reaction product, i.e., 2',7'-dichlorofluorescein, was measuredasdescribedabove. In eachsample,l X lo4 cells were analyzed; the results are expressed as the mean of the fluorescence intensity. Measurement of NADPH oxidase activity in a cell-free system

NADPHoxidase activity wasmeasured by means of a Clark oxygen-electrode at 27°C asthe rate of oxygen consumption by the cytosolic and membranous componentsof the NADPH oxidase thatwere isolated from neutrophils as described (24). In short, 400 1.11 of 10 mM HEPES containing 0.17 M sucrose, 75 mM NaCI, 0.5 mM EGTA, 1

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SURFACTANT SUPPRESSES MONOCYTEBACTERICIDALFUNCTIONS

0.01

0.1

1

10

FITC-Surfactant (mM)

0

5

10

15

hr

FIGURE 1. Fluorescenceintensity of monocytesandgranulocytesincubatedwithFITC-surfactant.Monocytes (0) and granulocytes (0) were incubated with various concentrationsof FITC-surfactant for 30 rnin ( a )or with 0.4 mM FITC-surfactant for various periods(b)at 37°C. Excess FITC-surfactant was removedby washing three times; the fluorescence intensity was then measured with a FACStar. Results are expressed as the meanof the fluorescence intensity; a representative of three experiments is shown.

mM MgC12,2 mM NaN, (pH 7.0), and 10 yM GTPyS were mixed with 10 pl of membrane components and 10 yl of cytosol components, which are the equivalents of 1 X lo6 neutrophils. Assembly of the NADPH oxidase was initiated by the addition of SDS at a final concentration of 100 pM, and the reaction vessel was closed. After 3 min, NADPH was added at a final concentration of 250 pM, and oxygen consumption was measured next (24). The results are expressed as pM Odmin. Statistics

All results are the means and SD of at least three experiments. The significance of differences was determined with the Wilcoxon signed ranks test.

Results Uptake of FITC-surfactant by monocytes and granulocytes

To .determine whether monocytes or granulocytes ingest surfactant, this material waslabeled with FITC.Monocytes incubated with FITC-surfactant showed an increase in fluorescence dependent on the concentration of FITCsurfactant and the incubation period (Fig. 1). Granulocytes didnotbindor ingest detectable amounts of FITCsurfactant (Fig. 1). Since flow cytometry does not discern between FITC-surfactant inside the cells and on the cell surface, the localization of FITC-surfactant in monocytes was investigated by confocal laser scanning fluorescence microscopy. The results revealed that virtually all FITCsurfactant was localized inside the monocytes that were incubated with this material (Fig. 2 ) . The uptake of surfactant was confirmed by transmission electron microscopy (results not shown). Effect of surfactant o n the phagocytosis of S. aureus by monocytes

After incubation of monocytes with various concentrations of surfactant, the phagocytosis of S. aureus at 60 min was

decreased ( p < 0.03) in a dose-dependent way, as determined microbiologically (Fig. 3a). Microscopic assessment of phagocytosis, using lysostaphin to eliminate extracellular S. aureus, also revealed that significantly ( p < 0.03) fewer bacteria were ingested by surfactant-incubated monocytes than by control monocytes, but that the percentage of bacteria adherent to the surface of surfactantincubated monocytes and control monocytes did notdiffer ( p > 0.2). Effect of surfactant on the intracellular killing of

S. aureus by monocytes

Intracellular killing of pre-opsonized S. aureus by monocytes incubated with surfactant was decreased compared to that by control monocytes (Table I and Fig. 36). Concentrations of surfactant as low as 1 mM caused significantly ( p < 0.05) reduced killing of S. aureus by monocytes, whereas 4 and 8 mM surfactant almost completely prevented the killing of ingested S. aureus (Fig. 36). On the basis of these results, 4 mM surfactant was used in all additional experiments. Incubation of monocytes with surfactant for periods shorter than 30 min also affected the intracellular killing of S. aureus: incubation for 1 and 5 min yielded killing values of 33 -t 17% and 22 -+ 25% ( n = 3), respectively, whereas the intracellular killing of S. aureus by control monocytes incubated with saline for these intervals was not affected. Since the effects of incubation with surfactant for 30 and 60 min were similar (intracellular killing values 19 -C 32% and 17 2 25%, respectively, n = 3), a 30-min period was used in subsequent experiments. To investigate whether impairment of the intracellular killing of S. aureus by monocytes incubated withsurfactant is reversible, monocytes were incubated with 4 mM surfactant for 30 min at 37"C, washed four times in PBS, and cultured for various periods in medium without surfactant at 37°C. The intracellular killing of S. aureus by surfactantincubated monocytes cultured for 0, 1, 2 , or 3 days

journal of Immunology

2395

FIGURE 2. Ingestion of FITC-surfactant by monocytes assessed by confocal laser scanning fluorescence microscopy. Monocytes were incubated with 4 mM FITC-surfactant for 30 min at 37°C. Excess FITC-surfactant was removed by washing three times; the cells were then stainedfor the cell-surfaceAg CD14 with biotinylated horse-anti-mouseIgG and streptavidin-labeled phycoerythrin and Texas red. Dual-wavelength images were obtained with a confocal laser scanning fluorescencemicroscope at an excitation wavelength of 488 nm, a short pass 530-nm filter, and a long pass 630-nm filter. Bar, 5 pm.

amounted to 13 t 17%, 45 t 9%, 21 ? 17%. and 6? 10% ( n = 3), respectively, all of which are significantly ( p < 0.05) less than the respective values (66 ? 13%, 81 -t 6%, 69 2 11%, and 50 2 15%; n = 3) found for control monocytes incubated with saline.These results demonstrate that the surfactant-induced impairment of the monocyte antibacterial functions is irreversible. Since the intracellular killing of bacteria by monocytes requires continuous membrane stimulationby serum components (25), we investigated whether surfactant inhibits the interaction between serum components and the corresponding receptors on monocytes. When surfactant was added at various times after the initiation of intracellular killing by serum, the intracellular killing of S. aureus was not affected during the first 10 to 15 min; subsequently, however, the number of intracellular bacteria did not decrease but increased (results not shown). Since sheep surfactant and human monocytes were used in most ofthe experiments, the effect of human surfactant on the intracellular killing of S. aureus by human monocytes and also the effect of sheep surfactant on the killing of S. aureus by sheep monocytes were investigated. The intracellular killing of S. aureus by human monocytes incubated with human surfactantwas significantly( p < 0.02) reduced comparedto that foundfor control monocytes (respective values 10 ? 14%and 59 ? IO%, n = 4). The moderate killing of S. aureus by sheep monocytes (31%) was completely inhibited by incubation of sheep monocytes

with sheep surfactant. The inhibitory effect of surfactant on the intracellular killing of S. aureus by monocytes was not due to LPS contamination of surfactant, since incubation of monocytes with 3 ng LPS/ml (equivalent to the concentration of LPS found in 4mM surfactant) did not affect the killing of S. aureus by these cells (data not shown). Since bacteria invading the alveoli also come into contact with surfactant, the phagocytosis intracellular and killing of surfactant-coated S. aureus by monocytes wereinvestigated. The phagocytosis of pre-opsonized surfactant-coated and pre-opsonized control S. aureus did not differ, as determined by microscopy using lysostaphin to eliminate adherent S.aureus (data not shown).The intracellular killing of surfactant-coated bacteria by normal monocytes was significantly impaired comparedto that of of control bacteria (18 -t 21% and 66 ? 18%, respectively, n = 5). Effect of surfactant on the phagocytosis and intracellular killing of other bacteria by monocytes

Monocytes incubated with 4mM surfactant exhibited significantly decreased ( p < 0.05) phagocytosis of S. pneumoniae 69 ? 9% (control cells 86 ? 5%) and E. coli 72 2 10% (control cells 82 ? 10%) ( n = 4). Surfactant did not affect the in vitro growth of these bacteria (data not shown). The phagocytosis of S. epidermidis and S. pyogenes by monocytes was not affectedsurfactant by (data not shown). The intracellular killing of S. pneumoniae, S. pyo-

2396


:I

100

I

2

0

1

T T

4

8

0

Surfactant (mM)

2

1

4

8

Surfactant (mM)

FIGURE 3. Effect of surfactant on the phagocytosis ( a ) andintracellular killing (b) of opsonized S. aureus by human monocytes. Monocytes were incubated with various concentrations of surfactant for 3 0 min at 37°C. Control monocytes were incubated with saline. To determine phagocytosis, monocytes and S. aureus were incubated at a ratio of 1 :1 in the presence of AB serum ( n = 6 experiments). To determine intracellular killing, monocytes and pre-opsonized S. aureus were incubated at a ratio of 1 : l ; after 3 min of phagocytosis, extracellular bacteria were removed by washing, and killing of the ingested bacteria was initiated by the addition of AB serum ( n = 4 experiments). Results are expressed as the percentage phagocytosis or intracellular killing at 60 min. Table I Effect of surfactant on the intracellular killing of S. aureus, S. epidermidis, E. coli, 5. pyogenes, and S. pneumoniae by human monocytes" 5. pyogenes

(%I 0 4 "

9062 f 14 18 2 19'

*

2 72 2 11

94 2 3 78 -t 10'

71 2 1 7 57 r 15

*

86 9 69 f g C

Determined as the percentage decrease in the number of viable Intracellular bacterla at 60 min ( n = 4). Final concentration of surfactant expressed as mM organic phosphate. p < 0.05 for the difference between monocytes incubated with surfactant and control rnonocvtes.

genes, S. epidermidis, and E. coli by monocytes incubated with 4 mM surfactant was decreased compared to that by control monocytes (Table I). Effect of DPI on the intracellular killing of bacteria by monocytes

The NADPH oxidase inhibitor DPI significantly ( p < 0.05) inhibited the intracellular killing of S. aureus, S. epidermidis, and E. coli, but not S.pyogenes by monocytes (Table 11). Phagocytosis and intracellular killing of S. aureus by granulocytes incubated with surfactant

Incubation of human granulocytes with 4 or 8 mM surfactant for 30 min did not affect either the phagocytosisor the intracellular killing of S. uureus (Table 111). The intracellular killing of S. aureus also was not affected when granulocytes were incubated with surfactant for 1 or 10 min (intracellular killing 93 2 5% and 91 2 6%,respectively, n = 6).

Becausegranulocytes do not ingest surfactant, it was asked whether the capacity of granulocytes to kill bacteria intracellularly is affected when surfactant is present inside these cells. Therefore, pre-opsonizedS. aureus were coated with surfactant, and these bacteria were used to introduce surfactantintogranulocytes. The phagocytosis of preopsonized surfactant-coated and pre-opsonized control S. uureus by granulocytes did not differ, when assessed microscopically using lysostaphin (data not shown). The intracellular killing of pre-opsonized surfactant-coated s.uureus by granulocytes in the absence of extracellular serum was significantlyimpairedcomparedto that of preopsonized control bacteria (28 2 14% and 78 t 6 8 , respectively, n = 4), indicating thatthe presence of surfactant within granulocytes leads to impaired killing of bacteria.

H2O2 release by monocytes and granulocytes incubated with surfactant To investigate whether the decreasedkilling of bacteria by monocytes after incubation with surfactant was due to im-

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Table I I Effect of DPI on the intracellular killing of S. aureus, S. epidermidis, E. coli, and S. pyogenes by monocytes' DPI

S. aureus

S. epidermidis

(PM)

(%)

(%)

0 5

76f3 1 5 2 12h

75 f 10 52f lob

E. coli (%I

S. pyogenes

74 f 20 48f34b

77 f 9 63f10

(%)

Determined as the percentagedecrease in the number of viable iniracellular and cell-associated bacteria at 30 min ( n = 4). p < 0.05 for the difference between monocytes incubated with DPI and control monocytes.

Table 111 Effect of various concentrations of surfactant on the phagocytosis and intracellular killing of S. aureus by human granulocytes Surfactant (mM)

0 4 8

Phagocytosis" ('in)

77 f a 75 f 9 76 f 7

77 f 16 84 f 13 8 6 f 11

a Determined as the percentage decrease in the number of viable extracellular bacteria at 30 min ( n = 3). Determined as the percentage decrease in the number of viable intracellular bacteria at 60 min ( n = 3 ) .

paired oxygen-dependentbactericidalmechanisms, we measured the release of H202 by surfactant-incubated monocytes and granulocytes upon stimulation with PMA. Monocytesincubated for 30 minwith 4 mM surfactant showed significantly ( p < 0.01) decreased H202 release upon stimulation with 5 ng PMNml but not after stimulation with25 ng PMA/ml (Table IVj. After incubation with 0.4 mM surfactant for 20 h, monocytes exhibited a significant ( p < 0.05) inhibition of their release of H2O2 upon stimulation with 5 and 25 ng PMNml (Table IV). Granulocytes incubated for 30 min with 4 mM surfactant displayed a reducedH202 release upon stimulationwith 1 and 5 ng PMA/ml but not after stimulation with2.5 ng PMNml; incubation of these cells for 20 h with 0.4 mM surfactant did not result in a decreased H202 release upon stimulation with 1 and 25 ng PMA/ml (Table IV).

H202 compared to control cells (Fig. 4). However, granulocytes that had ingested pre-opsonized surfactant-coated S. aureus did not exhibit ( p > 0.05) enhanced intracellular production of H202 upon stimulation with PMA, whereas the intracellular production of H202 by granulocytes that had ingested pre-opsonized control S. aureus was significantly ( p < 0.02) enhanced upon stimulation with PMA (Fig. 5). Effect of surfactant on the assembly of the NADPH oxidase

The NADPH oxidase consists of cytoplasmic and membranouscomponents;afteraddition of astimulus, these components assemble toform a functional enzyme by which electrons are transferredfrom NADPH to molecular oxygen (26). A cell-free system was used to investigate whether surfactant impairs the assembly of the components of the NADPH oxidase or the transfer of electrons. Oxygen consumption by the NADPH oxidase complex was inhibited by sheep as well as human surfactant in a concentration-dependent fashion when this material was added to a mixture of the components of the NADPH oxidase before addition of SDS, i.e., before assembly of the components into a functionalenzyme complex (Fig.6). The possibility that inhibition of oxygen consumption by surfactant was due to interference with the electron transfer was excluded by adding surfactant to the system after assembly of the various components into an active NADPH oxidase had occurred. The respectivevalues before and after addition of 0.4 mM sheep surfactant were 13 2 2.1 pM/min and 11 i 1.6 pM/min. Antibacterial functions of alveolar macrophages

Human alveolar macrophages displayed efficient phagocytosis (83 ? 18%, n = 4) but limited killing of ingested S. aureus (14 2 21%, n = 8). H202release upon stimulation of these cells with 25 ng PMA/ml was low ( 3 t 1.8 nmol/ ( lo6cells X 60 rnin), n = 5 ) compared to thatby monocytes (Table IV).

Intracellular production of H 2 0 2by monocytes and granulocytes incubated with surfactant

Since surfactant inhibits the intracellular killing of S. aureus inside phagocytes, it was essential to investigate the effect of surfactant on the intracellular productionof H202 by monocytes and granulocytes. The results revealed that surfactant abolishedthe production of intracellular H202 in monocytes stimulated with 100 ng PMA/ml (Fig. 4). The loading of controlmonocytesandmonocytesincubated with surfactant with DCFH-DA was comparable, as measured spectrofluorometrically, in the lysates of these cells after the addition of H202 (data not shown). Granulocytesincubated with surfactant were not impaired in theirPMA-inducedintracellularproduction of

Discussion The main conclusion to be drawn from the present results is that surfactantwhen located within monocytes and granulocytes inhibits the intracellular killingof bacteria by impairing oxygen-dependent killing mechanisms through inhibition of the assembly of components of the NADPH oxidase. This conclusion is based on the following arguments. 1) Monocytes incubated with surfactant ingest this material. 2) These cells display an impaired intracellular production of H202upon stimulation with PMA. 3 ) Granulocytes do not ingest surfactant and their capacity to kill bacteria or to produce H202 intraceliularly is not affected

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Table I V Effect of surfactant on the hydrogen peroxide release by monocytes and granulocytes H 2 0 2Release by Granulocytes

Release by Monocytes upon witha Stimulation Stimulation upon with"

H202

of

Time Incubation of lncubat~on Cells with

5PMNml ng

Saline 4 rnM surfactant

30 min 30 rnin

Saline 0.4 rnM surfactant

20 h 20 h

13 8

* 2.7 * 3.6'

7 2 1.6 4

1PMAfinl ng

25PMAfml ng

* 0.3

5PMAfml ng

*

13 2 2.8 14 f 2.1

9

11 2 1.7 8 f 2.3'

6 f 4.5 5 2.2

3.9 3 f 1.3b

17 12

25 ng PMAfrnl

* 2.3 * 4.0b

17 15

ND"

*

* 3.1 * 3.2

15 f 7.0 16 6.3

*

NDC

Results are expressed in nmol/(lOb cells x 60 min), n = 3 to 6. " p < 0.05 for the difference between cells incubated with surfactant and the remective control. ND, not done. 1)

30

El

0

0

15

30

min

0

15

30

rnin

FIGURE 4. Effect of surfactant on the intracellular H202production in monocytes ( a ) and granulocytes (b)upon stimulation for 30 min at 37°C. After with PMA. Monocytes and granulocytes were incubated with 4 mM surfactant (a)or saline (0) loading thecells with DCFH-DA,PMA (100 ng/ml) was added as a stimulus. Fluorescence intensity, as a measure of intracellular H 2 0 2 production, was determined with a FACStar. Results are expressedas the mean of the fluorescence intensity; a representative of three experiments is shown.

after incubation with surfactant.4) The introduction of surfactant into granulocytes, by allowing these cells to ingest pre-opsonizedsurfactant-coated S. aureus, leadsto both impaired killing of these bacteria and intracellular production of H202. 5 ) Surfactant reduces the oxygen consumption by the NADPH oxidase by inhibiting the assembly of cytoplasmic and membranous components into a functional enzyme. The mechanism for the inhibition of the assembly of the NADPH oxidase by surfactant is unknown. To justify the conclusion that intracellular surfactant inhibits the bactericidal functions of phagocytes, it was crucial to demonstrate the exact localization of FITCsurfactant in monocytes and granulocytes incubated with this material. By means of confocal laser scanning fluorescence microscopy, it could be demonstrated that FITCsurfactant is ingested by humanmonocytesbutnot by granulocytes. Studies with transmission electron microscopy confirmed the intracellular localization of surfactant in monocytes. For practical reasons, sheep surfactant insteadof human surfactant was used in this study. There are no major differences in the composition of sheep and human surfactant ( 16, 17). Comparable results werefound when human

monocytes were incubated with surfactant of sheep and of sheepmonocytes with humanorigin,andincubation sheep surfactant also resulted in complete impairment of their capacity to kill ingested S. aureus. Sheep and human surfactant were also equally effective in inhibiting the assembly of the NADPH oxidasein a cell-free system. Therefore, we concluded that it was justifiable to use sheep surfactantandhumanphagocytes.Itislikelythatthe concentration of surfactant used in the presentstudyis similar to that encountered by monocytes when they enter the alveoli, because the ratio between the amount of surfactant and the number of phagocytes used in the present study was comparable to such ratios calculated for the lungs of sheep (27), rabbits (2, 28), and rats (2, 29). The effect of surfactant on the antibacterial functions of monocytes could not be due to LPS contamination of surfactant, since incubation of monocytes with concentrations of LPS comparable to those found in surfactant did not affect the intracellular killing of S. aureus by monocytes. The results described in the present study are in agreement with our earlier finding thatthe bactericidal functions of mouse peritoneal macrophages are inhibited after incubation with sheep surfactant (3). It has also been reported

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Journal of Immunology 15

n

C

-

'5

I

10

3.

LG 30

15

C

.-c0

n E 3 1

8

5

c? 0 0.1

rnin

FIGURE 5. Effect of ingestionofpre-opsonized surfactantcoated s. aureus on the intracellular production of H 2 0 2by granulocytes upon stimulation withPMA. Granulocytes were or preallowed to ingest pre-opsonized surfactant-coated (1) s. aureus for 3 min at37°C. Nonopsonized control (0) ingested bacteriawereremoved by differentialcentrifugation, after which the cells were loaded with DCFH-DA and next stimulated with 100 ng PMNmI.Fluorescence intensity, as a measure for intracellular H 2 0 2production, was determined with a FACStar. Results are expressed as the percentage increase in fluorescence intensity at 15 and 30 min comfluorescence intensity at t = 0 ( n = 4 pared to the experiments).

that surfactant inhibits the killing of Candida albicans ( 5 ) and Streptococcus agalactiae (6) by newborn rabbit alveolar macrophages and impairs the PMA-or opsonized zymosan-induced chemiluminescence by rabbit alveolar macrophages (4). Surfactant also affects the functional activities of T lymphocytes (30, 3 1). It is important to realize that in vivo the functions of various types of cell localized in the alveoli are influenced by the presence of surfactant. Monocytes incubated with surfactant resemble alveolar macrophages as far as the intracellular presence of surfactant (1 l), their limited antibacterial functions (Ref. 8 and the present study), and their capacity to release H 2 0 2 upon stimulation with PMA (Refs. 8, 32, and the present study) are concerned. Granulocytes that are not affected by incubation with surfactant display impaired bactericidal functioning after ingestion of surfactant-coated bacteria. The question of the mechanisms responsible for the removal of bacteria-which may be coated with surfactant-from the alveoli remains unanswered. Bacteria ingested by alveolar macrophages, monocytes, and granulocytes leave the alveoli as a result of migration of these phagocytes to the upper parts of the respiratory tract and expulsion from the body via mucociliary movement and forced respiration. In addition to the inhibition of both intracellular killing of bacteria and the intracellular production of H202 by sur-

0.2

0.3

0.4

Surfactant (mM) FIGURE 6. Effect of surfactant on activation ofthe NADPH suroxidase in a cell-free system. Sheep (0)or human (0) factant was added to the membranous and cytosolic components of the NADPHoxidase before assembly of the complex had occurred. After a few minutes, SDS and NADPH were added and oxygen consumption was measured using a Clark electrode ( n = 3 experiments).

factant, another important finding was the reduced release of H202 by both monocytes and granulocytes incubated with surfactant. Since this material is not ingested by granulocytes, extracellular surfactant must account for the impaired H202 release resulting from inhibition of the assembly of the NADPH oxidase in the plasma membrane. These results could mean that in vivo surfactant plays an important role inthe protection of alveolar epithelium from the injury caused by reactive oxygen intermediates released by phagocytes.

Acknowledgments The authors thank Mrs. E. de Backer-Vledder and Mr. B. M. van de Berg for technical assistance, Mr. M. van der Keur and Mr. J. Slats (Department of Cytochemistry and Cytometry, University of Leiden, The Netherlands) for assistance with FACS analysis and confocal laser scanning fluorescence microscopy, Dr. G . Zetterberg (Department of Thoracic Medicine, Karolinsky Hospital, Stockholm, Sweden) for his advice on the labeling of surfactant with FITC, Dr. J. Stolk (Department of Pulmonary Diseases, University Hospital, Leiden, The Netherlands) for providing the human BAL fluid, and Dr. A. J. Verhoeven (Laboratory for Experimental and Clinical Immunology, University of Amsterdam, The Netherlands) for his help in measuring oxygen consumption in the cell-free system at his laboratory.

References 1. Fels, A. 0. S., and Z. A. Cohn. 1986. The alveolar macrophage. J. Appl. Physiol. 60:353. 2. Wright, J. R., and J. A. Clements. 1987. Metabolism and turnover of lung surfactant. Am. Rev. Respir: Dis. 135:426. 3. Nibbering, P. H., M. Th. van den Barselaar, J. S. van de Gevel, P. C . J. Leijh, and R. van Furth. 1989. Deficient intracellular

2400

4.

5.

6

7.

8.

9.

10.

11. 12.

13.

14. 15.

16.

17.

18.

19.


killing of bacteria by murine alveolar macrophages. Am. J. Respir: Cell Mol. Biol. 1:417. Hayakawa,H., Q. N. Myrvik, and R. W. St. Clair. 1989. Pulmonary surfactant inhibits priming of rabbit alveolar macrophages. Am. Rev. Respir: Dis. 140:1390. Zeligs, B. J., L. S. Nerurkar, and J. A. Bellanti. 1984. Chemotactic and candidacidal responses of rabbit alveolar macrophagesduringpostnataldevelopment and the modulating roles of surfactant in these responses. Infect. lmmun. 44:379. Sherman, M. P., J. B. D’Ambola, E. E. Aeberhard, and C. T. Barrett. 1988. Surfactant therapy of newborn rabbits impairs lung macrophage bactericidal activity. J. Appl. Physiol. 65: 137. Iwaarden, F. van, B. Welmers, J. Verhoef, H. P. Haagsman, and L. M. G. van Golde. 1990. Pulmonary surfactantenhances the host-defense mechanism of rat alveolar macrophages. Am. J. Respir: Cell Mol. Bid. 2.91. Hoidal, J. R., D. Schmeling, and P. K. Peterson. 1981. Phagocytosis, bacterial killing, and metabolism by purified human lung phagocytes. J. Infect. Dis. 144:61. BlussC van Oud Alblas, A,, and R. van Furth. 1979. Origin, kinetics, and characterization of pulmonary macrophages in the normal steady state. J. Exp. Med. 149:1504. BlussC van Oud Alblas,A,, H. Mattie, and R. van Furth. 1983. A quantitive evaluation of pulmonary macrophage kinetics. Cell Tissue Kirzet. 16:211. Nichols, B. A. 1976. Normal rabbit alveolar macrophages. J. Exp. Med. 144:906. BlussC van Oud Alblas, A,, B. van der Linden-Schrever, and R. van Furth. 198 1. Origin and kinetics of pulmonary macrophages during an inflammatory reaction induced by intra. 154: venous administration of heat-killed BCG. J. E , T ~Med. 235. Blusse van Oud Alblas, A,, B. van der Linden-Schrever, and R. van Furth. 1983. Origin and kinetics of pulmonary macrophages during an inflammatory reaction induced by intraalveolar administration of aerosolized heat-killed BCG. Am. Rev. Re.spiK Dis. 130:1177. Boyum, A. 1968. Separation of leucocytes from blood and bone marrow. Scur~d.J. Clin. Lab. Invest. 2/:[Suppl.] 21:7. Bartlett, G. R. 1959. Phosphorus assay in column chromatography. J. Biol. Chem. 234:466. Hallman, M., R. Spragg, J. H. Harrel, K. M. Moser, and L. Cluck. 1982. Evidence of lung surfactant abnormality in respiratory failure. Study of bronchoalveolar lavage phospholipids,surface activity, phospholipase activity, andplasma myoinositol. J. Clin. Invest. 70:673. Ikegami, M., A. Jobe, and T. Glatz. 1981. Surfactant activity following natural surfactant treatment in premature lambs. J. Appl. Physiol. 5/:306. Ugolini V., G. Nunez, R. G. Smith, P. Stastny, and J. D. Capra. 1980. Initial characterization of monoclonal antibodies against human monocytes. Proc. Nail. Acud. Sci. USA 77: 6 764. Furth, R. van, Th. L. van Zwet, and P. C. J. Leijh. 1978. In vitro determination of phagocytosis and intracellular killing

20.

by polymorphonuclear and mononuclearphagocytes. In Hundbook of Experimeniul Immunology, Ch. 32, D. M. Weir, ed. Blackwell ScientificPublications,Oxford. Bra&, p. J. van den, L. F. M. Buys, and B. M. P. Aleman. 1990. The antibacterial activity of benzylpenicillin against Stuphylococcus uureus ingested by granulocytes. J. Antimicrob. Chemother: 25:931. Ellis, J. A,, S. J. Mayer, and 0.T. G. Jones. 1988. The effect of the NADPH oxidase inhibitor diphenyleneiodonium on aerobic and anaerobic microbicidal activities of human neutrophils. Biochem. J. 251:887. Ruch, W., P. H. Cooper, and M. Baggiolini. 1983. Assay of H202 production by macrophages and neutrophils with homovanillicacidand horse-radish peroxidase. J. lmmunol. Methods 63:347. Bass, D. A., J. W. Parce, L. R. Dechatelet, P. Szejda, M. C. Seeds, and M. Thomas. 1983. Flow cytometricstudies of oxidativeproductformation by neutrophils: a graded response to membrane stimulation. J. Immunol. 130:19/0. Bolscher, B. G. J. M., S. W. Denis, A. J. Verhoeven, and D. Roos. 1990. The activity of one soluble component of the cell-free NADPH-0, oxidoreductase of human neutrophils depends on 5’-0-(3-thio)triphosphate. J. B i d Chem. 265:15782. Leijh, P. C. J., M. Th. van den Barselaar, T. L. van Zwet, M. R. Daha, and R. van Furth. 1979. Requirement of extracellular complement and immunoglobulin for intracellular killing of micro-organisms by human monocytes. J. Clin. Invest. 63: 772. Bromberg, Y., and E. Pick. 1985. Activation of NADPHdependent superoxide production in a cell-free systemby sodium dodecyl sulfate. J. Biol. Chem. 260:13539. Warner, A. E., B. E. Barry, and J. D. Brain. 1986. Pulmonary intravascular macrophages in sheep. Morphology and function of a novel constituent of the mononuclear phagocyte system. Lcth. Invest. 56:276. Zeligs, B. J., L. S. Nerurkar, and J. A. Bellanti. 1977. Maturation of the rabbit alveolar macrophage during animal development. l. Perinatal influx and ultrastructural differentiation. Pediutr: Res. 11:197. Masse, R., P. Fritsch, D. Nolibk, J. Lafuma, and J. Chretien. 1977. Cytokinetic study of alveolar macrophage renewal in rat. ERDA Symposium Series 43:106. Wilsher, M. L.. D. A. Hughes, and P. L. Haslam. 1988. Immunoregulatory properties of pulmonary surfactant: effect of lung lining fluid on proliferation of human blood lymphocytes. Thorax 43:354. Shimizu, M., B. Vayuvegula, M. Ellis, L. Cluck, and S. Gupta. 1988. Regulation of immune functions by human surfactant. Ann. Allergy 6/:459. Murray, H. W., R. A. Gellene, D. M. Libby, C. D. Rothermel, and B. Y. Rubin. 1985.Activation of tissue macrophages from AIDS patients: in vitro response of AIDS alveolar macroJ. Itnmunol. 135: phages to lymphokines and Interferon-y. 2374. ”

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

3I .

32.