Surfactant protein A modulates release of reactive oxygen species from alveolar macrophages

Surfactant protein A modulates release of reactive oxygen species from alveolar macrophages

STEFAN WEISSBACH, ANDREA NEUENDANK, MARGARITA PETTERSSON, TOM SCHABERG, AND ULRICH PISON Department of Anesthesiology and Intensive Care Medicine, UniversitEtsklinikum Rudolf Virchow, and Department of Infectious Diseases and Immunology, Chest Clinic Heckeshorn, Freie lJniversit&t Berlin, 13344 Berlin, Germany

Weissbach, Stefan, Andrea Neuendank, Margarita Pettersson, Tom Schaberg, and Ulrich Pison. Surfactant protein A modulates release of reactive oxygen species from alveolar macrophages. Am. J. Physiol. 267 (Lung CeZZ. Mol. Physiol. 11): L660-L666, 1994.-The production and release of reactive oxygen species (the respiratory burst) is a common metabolic pathway linked to several macrophage-related reactions. The most abundant surfactant protein A (SP-A) binds to alveolar macrophages (AM) through a specific surface receptor with high affinity. Because such binding might initiate or modulate the respiratory burst, we wanted to know whether and how SP-A affects the oxygen radical release from AM. To answer these questions, we measured the release of reactive oxygen species from rat AM under various in vitro conditions using enhanced chemiluminescence systems. We prepared SP-A from pulmonary surfactant isolated either from silica- treated rats or adult dogs Resident AM were harvested from pathogen-free Wistar rats by lung lavage. Adhered and nonadhered AM were assessed on protein-free or protein-coated surfaces of 96-well microtiter plates. On protein-free surfaces, the sole addition of SP-A failed to induce measurable oxygen radical release from 2 x lo5 adhered or nonadhered AM, while zymosan opsonized with SP-A induced a marked increase over control. On protein-coated surfaces, AM respond differently depending on the coated protein: on SP-A-coated surfaces, a dose-dependent enhancement of oxygen radical release with a mean effective concentration of - 1.15 pg/ml was found. No such enhancement was seen on plates coated with similar amounts of either human fibronectin or collagen, and the enhancement with serum albumin was not dose related. Our data demonstrate that SP-A only enhances oxygen radical release from AM if SP-A is fixed to zymosan or the surface of the reaction vial in vitro. We conclude that SP-A surface interactions are required to release oxygen radicals from AM in vitro. We speculate that a multivalent interaction of SP-A with macrophages may be necessary for a functional response of AM in vitro, and a comparable mechanism might take place in modulating host defense reactions of AM in vivo.

pulmonary surfactants; apoproteins; C-type lectin; respiratory host defenses

THE PREDOMINANT CELL TYPE in the alveolar compart- ment of the lungs under normal conditions are alveolar macrophages (6, 10). The alveolar macrophage coexists with pulmonary surfactant in the liquid lining layer that covers the alveolar surface (32, 33, 41). Pulmonary surfactant is a complex mixture of different components: phospholipids, few other lipids, and the four genetically distinct surfactant proteins (SP) SP-A, SP-B, SP-C, and SP-D (3,12,27). Although the major function of pulmonary surfactant is maintaining decreased surface tension in the alveoli at decreased lung volumes (9),

surfactant may also contribute to endogenous defense mechanisms of the lungs (see Ref. 24 for review). Many defense mechanisms of the lungs require alveolar macrophages. We recently showed that the most abundant surfactant protein, SP-A, reacts with alveolar macrophages through a specific surface receptor (26). SP-A also stimulates chemotaxis of alveolar macrophages (42).

The aim of the present study was to test the hypothesis whether SP-A binding to macrophages would acti- vate this cell type. One measure of macrophage cell activation is the release of reactive oxygen species (respiratory or oxidative burst), which could be detected in vitro using enhanced chemiluminescence. Hence, to assess the influence of SP-A on alveolar macrophages, we performed experiments to characterize whether and how SP-A affects the oxygen radical release from alveolar macrophages using such enhanced chemiluminescence systems.

MATERIALS AND METHODS

CeZZ preparation. Alveolar macrophages were harvested by lavaging the lungs of pathogen-free Wistar rats (250 g). We lavaged rats with lo-ml aliquots of a buffer containing (in mM) 140 NaCl, 5 KCl, 2.5 sodium phosphate buffer, 10 mM IV-Z- hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), 6 glucose, 0.2 ethylene glycol-bis(P-aminoethyl ether)-N,N,N- tetraacetic acids (EGTA), pH 7.4, until a total volume of 100 ml buffer per rat was recovered. The lavage fluids from 2-3 animals were pooled and centrifuged (180 g, 10 min., 4”C), to pellet the cells. Cells were counted in a modified Neubauer chamber and then resuspended in Hanks’ balanced salt solution (HBSS) containing 0.1% (wt/vol> gelatin at a final concentration of 4 x lo6 cells/ml. Peripheral monocytes were prepared from rat blood obtained by cardiac puncture supplemented with heparin. After sedimentation for 1 h in 6% dextran in normal saline at 37°C a cell-rich plasma solution was obtained, transferred in a new tube containing Ficoll-Paque (1:6; vol/vol), and centrifuged for 40 min at 20°C and 400 g. The mononuclear cell layer at the interphase was removed and washed twice with HBSS containing 0.1% (wt/vol) gelatin. Cells were added to plastic petri dishes to separate adhering (monocytes) from nonadhering cells (lymphocytes). Nonad- hered cells were washed off the plates, adhered cells were removed and suspended in HBSS-gelatin at a final concentration of 4 x lo6 cells/ml. Viability of cells was evaluated by the trypan blue exclusion method. An aliquot of the cells was used to prepare cytocentrifuge slides for differential cell counts.

Surfactant and SP-A preparation. Pulmonary (whole) surfactant was isolated from lung lavage fluids using differential centrifugation techniques as described earlier (25). Lung lavages were obtained from adult dogs or rats given an intra- tracheal instillation of 0.5 ml isotonic saline containing 25 mg of silica 4 wk before lung lavage, according to the procedure described by Dethloff et al. (4). SP-A was prepared from whole

IA660 1040-0605/94 $3.00 Copyright o 1994 the American Physiological Society

SP-A, ALVEOLAR MACROPHAGES, AND OXYGEN RADICAL RELEASE L661

surfactant of either species by sequential extraction with butanol and n-octyl p-D-glucopyranoside as described (25). Purity of SP-A preparations was checked using 12% polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) (17). Protein concentration was determined as described by Lowry et al. (20) and modified by Lees and Paxman (19). Phospholipid concentrations of whole surfactant were calculated from the organic phosphorus content (2). SP-A purified from alveolar proteinosis patients was a gift of S. Haw- good, Cardiovascular Research Institute, San Francisco, CA.

Iodination of SP-A. Purified SP-A was iodinated by the lactoperoxidase-glucose oxidase procedure using Bio-Rad Enzy- mobeads as described previously (26). Free Na125 was sepa- rated from 1251-labeled protein on a Bio-Gel PGDG column. Fractions that contained radioactivity that were > 90% precipi- table by trichloroacetic acid were combined, stored at 4°C and used within a week. Specific activity ranged from 0.7 to 1.6 @i/ pg protein.

Endotoxin measurements. We attempted to measure gram- negative bacterial endotoxin contaminations in our cell and SP-A preparations using a lysate derived from washed Limu- Zus amebocytes of Limulus poZyphemus (Chromogenix, Moln- dal, Sweden). The assay was carried out according to manufac- turer’s instructions, and results were expressed as endotoxin units per milliliter (EU/ml), as suggested in the European Pharmacopoea ( 1).

Chemiluminescence assay. To characterize whether and how SP-A affects oxygen radical release from alveolar macrophages or peripheral monocytes, we used luminol- or lucigenin- enhanced chemiluminescence. We performed experiments in triplicates in 96-well microtiter plates (Dynatech Micro-Fluor, N-Plates, Dynatech, Denkendorf, Germany). Chemilumines- cence was measured using the Amerlite Research Luminom- eter (Amersham Buchler, Braunschweig, Germany). Photon emission per well was measured within 1 s and monitored continuously for all 96 wells for at least 60 min. Data were collected on-line, downloaded into a personal computer, and analyzed using Excel (Microsoft). Data are presented as percent- age of the corresponding buffer controls [% counts/min (cpm)], unless otherwise stated. For the assay, alveolar macrophages or peripheral monocytes in 50 ~1 gelatin-HBSS were incubated at a density of 200,000 cells per well at 37°C and 5% CO2 for 1 h on protein-free or protein-precoated plates to allow adherence, or were placed into the reaction vials immediately before stimulation for studying nonadhered cells. Then, 75 ~1 HBSS were added, followed by 100 ~1 luminol or lucigenin (2 x lop4 M final concentration, respectively), and then by 25 ~1 of either opsonized or nonopsonized zymosan (1 kg/ml final concentration), phorbol myristate acetate (PMA) (5 x lop7 M final concentration), whole surfactant or SP-A at various concentrations, or gelatin-HBSS, giving a final volume of 250 pi/well. Microtiter plates were shaken for 30 s and then immediately placed into the chemiluminometer to start measurements at 35°C.

Precoating. Microtiter plates were coated with various concentrations of rat or dog SP-A, human fibrinogen, bovine serum albumin, or collagen type IX from human placenta (Sigma, Munich, Germany). Coating was performed with the various substances in 0.1 M sodium carbonate buffer (pH 9.6) and by incubating the plates overnight at 4°C. Plates were washed three times with phosphate-buffered saline (pH 7.4) before being used in our chemiluminescence assay. Coating efficiency for SP-A was analyzed using the radiolabeled protein under the same conditions as the unlabeled protein. The amount of radioactivity coated to each vial of the plates was then measured in a gamma counter (Cobra II, Canberra- Packard, Frankfurt, Germany).

Opsonixation. Four milliliters of zymosan (2.5 mg/ml) in HBSS were opsonized with pooled human AB serum (1 ml), heat-inactivated serum (1 ml), or SP-A (40 pg). Zymosan incubated with HBSS served as nonopsonized control. After incubation for 30 min at 37°C we centrifuged the material and the pelleted zymosan was washed once in 12 ml HBSS and finally resuspended in 1 ml HBSS to give a final concentration of 10 mg/ml. Heat inactivation of serum was performed by incubation for 30 min at 56°C.

Statistics. All data are presented as means 2 SE. Compari- sons of data obtained with controls and various experimental settings were made using the paired t-test or by one-way analysis of variance for multiple comparisons. Correlation coefficients were calculated by linear regression analysis. Differences were considered significant at P < 0.05.

RESULTS

The alveolar cells obtained from pathogen-free rat lung washings were > 90% macrophages as determined by differential cell counts of cytocentrifuge preparations and had a viability of > 95%. We harvested 1 x lo6 to 3 x lo6 cells/rat. Peripheral monocyte preparations were 85-90% pure and had a viability of > 90%. Whole surfactant had a phospholipid-to-protein ratio of - 1O:l by weight. The protein fraction consisted of - 60% contaminating serum proteins, 30% SP-A, and 10% SP-B/SP-C. The recovery of purified SP-A was -0.3- 0.5 mg per rat that had silica instilled a month earlier and -2.5-5 mg per dog. Both SP-A preparations ap- peared very similar on SDS-polyacrylamide gels, differ- ing only in the relative composition of their respective triplets, which probably represents different glycosyla- tion (13). For comparison, SP-A purified from alveolar proteinosis patients showed only two bands, one at - 36 kDa, the other at 62 kDa (Fig. 1). The 62-kDa band probably represents nonreducible cross-linked beta chains of the alveolar proteinosis SP-A (36). We checked cell, surfactant, and SP-A preparations for possible endotoxin contaminations. No endotoxin contaminants could be detected down to sensitivities of 0.06 EU/ml.

To test the effect of SP-A on the lucigenin-enhanced production of reactive oxygen species, we added sole SP-A purified from dog or rat lung washings, zymosan opsonized with such SP-A, zymosan opsonized with pooled AB-serum, zymosan opsonized with heat-inactivated serum, or zymosan without any opsonization into the fluid phase of cell cultures containing alveolar macrophages. While the sole addition of SP-A (0.5-80 pg/ml) failed to produce more reactive oxygen species as control, zymosan opsonized with SP-A induced a marked increase over control, as did zymosan opsonized with AB-serum or heat-inactivated serum. The oxygen radical release induced with the various opsonized zymosans was always significantly higher than with buffer control and nonopsonized zymosan, with peak activities occurring between 10 and 20 min (Fig. 2). We found similar effects for SP-A prepared from dog or rat lung washings.

Because sole SP-A added into the fluid phase of alveolar macrophage cultures did not produce more reactive oxygen species compared with control, but zymosan opsonized with SP-A did, we hypothesized that surface binding of SP-A is important for the examined

L662 SP-A. ALVEOLAR MACROPHAGES. AND OXYGEN RADICAL RELEASE

Fig. 1. Surfactant protein A @P-A) preparations appear i differently under reduced conditions on a 12% polyacryl- ;’ amide gel. From left to right: molecular mass standards, SP-A from silica-treated rat, dog, and alveolar proteino- j sis patients. Molecular mass standards represent 106, ;. 80,49,27, and 18 kDa (from top to bottom).

biological reaction. To further test this hypothesis, we coated SP-A at various concentrations (5-80 p,g/ml) to the surface of the reaction vials and measured lucigenin- enhanced chemiluminescence. We found that surface- coated SP-A enhanced the release of reactive oxygen species dose-dependently, with peak activities occurring between 0 and 5 min for adhered macrophages (Fig. 3A) and peak activities at - 5-10 min for nonadhered cells (Fig. 3B). Cell adherence led to a significantly more pronounced response of the macrophages comparing corresponding maxima, except for the highest SP-A concentration (80 pg/ml), which revealed similar maxima for adhered and nonadhered cells. We tried to further stimulate macrophages by additionally adding serum- opsonized zymosan or PMA at fixed concentrations (1 pg/ml and 5 x lop7 M, respectively), or SP-A at different concentrations (5-80 pg/ml) to the fluid phase of the reaction vials, which were precoated with SP-A. With all three additional stimuli tested over the whole range of SP-A concentrations coated to the plates (5-80 pg/ml), we found no significant further increase of oxygen radical release (data not shown).

To calculate the amount of SP-A coated to the cell- culture plates, we radiolabeled SP-A and incubated the labeled protein as we did with the nonlabeled protein. We found that the plating efficiency was dependent on the protein concentration used, ranging between 7.5 and 20% with linearity in about one order of magnitude.

,

Using these binding data, we plotted l/effect vs. l/concentration to calculate the mean effective concentration (EC&) for SP-A releasing oxygen radicals. The EC& for SP-A to release oxygen radicals from alveolar macrophages was found at N 1.15 kg/ml, the maximum effect at 1,724 cpm (Fig. 4).

To examine whether other surface-coated proteins might modulate the production of reactive oxygen species, we coated human fibronectin, bovine serum albumin, or collagen in similar amounts as SP-A on microti- tre plates and measured the lucigenin-enhanced chemiluminescence induced by the coated proteins alone or together with serum-opsonized zymosan or PMA. None of the tested proteins showed the effects found for SP-A. Specifically, none of the coated proteins alone nor in conjunction with serum-opsonized zymosan or PMA showed dose-dependent effects similar to those found for SP-A, although albumin enhanced the release of oxygen radicals.

Using the chemiluminescence amplifier lucigenin, surface-coated SP-A increased the production of reactive oxygen species by alveolar macrophages in a dose- dependent fashion. Using the chemiluminescence amplifier luminol, surface-coated SP-A showed no such increase, although we observed a slight enhancement over control (Fig. 5). However, in luminol-enhanced systems, our alveolar macrophage preparations never responded as well as in lucigenin-enhanced systems.

SP-A, ALVEOLAR MACROPHAGES, P LND OXYGEN RADICAL RELEASE L663

-C ABa-zymosan

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+- SPA-zymosan

* zymosan

-t SPA

-0- control

01 11 13 11 11 8 1 ! I1

0 10 20 30 40 50 60

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Fig. 2. SP-A (SPA) alone does not enhance release of reactive oxygen species from alveolar macrophages but could substitute for serum as an opsonin. While sole addition of 0.5430 pg SP-A failed to release more reactive oxygen species compared with control, zymosan opsonized with SP-A (SPA-zymosan) induced a marked increase over control, as does zymosan opsonized with AB-serum (ABa-zymosan) or heat-inactivated serum (ABi-zymosan). All opsonized zymosans were significantly more effective than the nonopsonized zymosan (zymo- San). SPA-zymosan is not significantly different from ABi-zymosan. We found similar effects for SP-A prepared from dog or rat lung washings. Depicted data are means 2 SE of 5 independent experiments, each done in triplicate, using rat SP-A and lucigenin as chemiluminescence amplifier. cpm, counts/min.

Surface-coated SP-A induced a dose-dependent release of reactive oxygen species from alveolar macrophages. In contrast, peripheral monocytes, at the same cell density as macrophages and with surface-coated

A B lOOO-

900 -

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600 - T &

or 1, 11 1 I I1 11 I1 I 0 10 20 time3:min] 40 50 60

lOOO-

900-

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SP-A at concentrations from 5 to 80 kg/ml, did not release detectable amounts of reactive oxygen species in our lucigenin-enhanced chemiluminescence assay.

With whole surfactant in solution (0.1-10 mgphospho- lipids) we found inhibition of the chemiluminescence response of adhered macrophages. The inhibition was - 25% compared with control and was more pronounced for higher phospholipid concentrations.

DISCUSSION

The most abundant surfactant protein, SP-A, modulates the release of reactive oxygen species from alveolar macrophages, a measure for cell activation in vitro. SP-A does not simply enhance the release of reactive oxygen species from these alveolar lining cells, which subsequently might result in unrestricted alveolar inflammation. Only SP-A coated to a surface such as zymosan or the reaction vial enhances dose-dependently oxygen radical release. Our observations indicate that either a multivalent interaction of SP-A with the macrophage surface is crucial for a cellular response or that a conformational change of the protein that is stabilized by immobilization is required for functionally engaging the receptor. Comparable results have been reported recently for the interaction of Clq, which is structurally homologous to SP-A, with neutrophils (11).

Our data show that SP-A surface interactions are required to release measurable amounts of oxygen radicals from alveolar macrophages in vitro, whereas the presence of pure SP-A in the fluid phase of macrophage cultures does not have this effect. The latter might reflect a normal environmental situation in alveoli where cell activation would be harmful to the host. Weber and co-workers (40) have shown that SP-A prepared from dog lung washings and added to the fluid phase of macrophage culture dishes was even able to inhibit the release of oxygen radicals under selected in

+ 8Opg/ml SPA

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d?r lOpg/ml SPA

-O- !ipg/ml SPA

U control

Yr 0 ’ ’ IO

T ’ 20 ’ ‘30’ ‘40’ ’ 50 ’

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Fig. 3. Surface-coated SP-A enhances release of reactive oxygen species in a dose-dependent manner. SP-A was coated onto surface of reaction vials at various concentrations, and we measured lucigenin-enhanced chemiluminescence as described in MATERIALS AND METHODS. Depicted data are means + SE of 5 independent experiments each done in triplicate, using rat SP-A and lucigenin as chemiluminescence amplifier and adhered (A) or nonadhered macrophages (B).

L664 SP-A, ALVEOLAR MACROPHAGES, AND OXYGEN RADICAL RELEASE

0.003 1

Fig. 4. Plot of l/effect (cpm) vs. l/concentration showing -l/half- maximal effective concentration (- 1 /EC& (intersection of slope with x-axis) and 1 / rnax,fect (intersection of slope with y-axis) for surface- coated SP-A releasing oxygen radicals. Equation for slope is y = 5.7813 x lop4 + 6.6113 x 10e4 and has a correlation coefficient of 0.991. Plot was drawn from data found by using rat SP-A and adhered macrophages (see Fig. 3A), maxeffect, maximum effect.

vitro conditions. These results were confirmed in a study by Katsura et al. (16), who measured superoxide production and found inhibition caused by rat SP-A. Their and our data, however, are in contrast to the findings of van Iwaarden et al. (34). They have shown that SP-A enhances the release of oxygen radicals from alveolar macrophages. Their SP-A, however, was purified from patients who had alveolar proteinosis. SP-A obtained from alveolar proteinosis patients differs from

1

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-A- 20pg/ml SPA

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-c- control

0 10 ' 20 30 40

time [min] ' 50 60

Fig. 5. Surface-coated SP-A has little effect on release of reactive oxygen species from alveolar macrophages in luminol-enhanced systems. SP-A was coated onto surface of reaction vials at various concentrations, and we measured luminol-enhanced chemiluminescence as described in MATERIALS AND METHODS. Depicted data are means + SE of 5 independent experiments, each done in triplicates, using rat SP-A and luminol as chemiluminescence amplifier.

SP-A prepared from healthy mammals: it appears differently on SDS-polyacrylamide gels (see Fig. l), has a different primary structure (36), a less ordered three- dimensional structure (39, and reacts differently with macrophages regarding the octadecameric superstruc- ture of the molecule (21). In addition, enzymatic break- down under pathological conditions might change SP-A as well (25), and substances that are toxic to cells may be present in washings from diseased lungs and contami- nate such SP-A preparations. We checked our SP-A and cell preparations for endotoxin contaminants and found no such contamination. Furthermore, different SP-A preparation techniques might change the protein’s structure, resulting in functional variation. Although our data did not support the simple conclusion that SP-A enhances chemiluminescence, it well supports the view that SP-A modulates macrophage activation. In addition, we would speculate that the conflicting data pub- lished by van Iwaarden’s group (34) might in fact demonstrate one possible pathogenic mechanism in alveolar proteinosis patients, revealing the need for further studies.

In contrast to the SP-A effects on alveolar macrophages, SP-A does not release measurable amounts of oxygen radical species from peripheral monocytes in our study. This finding indicate SP-A specificity for alveolar macrophages. Goodman and Tenner (11) showed that SP-A (from alveolar proteinosis patients) coated to microtiter plates in a similar way as in our study does not induce measurable amounts of superoxide production from neutrophils. SP-A, however, was able to enhance FcR- and CRl-mediated phagocytosis of, both, peripheral monocytes and culture-derived macrophages (30).

As a first step toward determining the metabolic pathway(s) involved during SP-A-mediated release of reactive oxygen species from alveolar macrophages, we compared lucigenin and luminol as amplifier in our chemiluminescence assay. Lucigenin and luminol appear to amplify chemiluminescence by different mechanisms and have different specificity for oxygen radicals (7, 38). 1) Lucigenin-amplified chemiluminescence can be generated in a cell-free xanthine/xanthine oxidase system by the reaction of superoxide anion radicals. This reaction is independent of myeloperoxidase but could be abolished by superoxide dismutase. 2) Luminol- amplified chemiluminescence can be generated in a cell-free system by the reaction of hydrogen peroxide with myeloperoxidase and a halide, and could be abolished by hydrogen scavengers such as catalase. Using the chemiluminescence amplifier lucigenin, surface- coated SP-A increased the production of reactive oxygen species by alveolar macrophages in a dose-dependent fashion. Using the chemiluminescence amplifier luminol, SP-A showed no such increase, although we observed a slight enhancement over control. These data suggest that surface-coated SP-A modulates the release of superoxide anion radicals probably through a myeloperoxidase-independent pathway. Further studies are needed to elucidate SP-A-related cell and membrane activating processes, i.e., with the use of inhibitors for various relevant metabolic pathways.

SP-A, ALVEOLAR MACROPHAGES, AND OXYGEN RADICAL RELEASE L665

Alveolar macrophages have been shown by immuno- histochemistry to contain SP-A (37) and to bind this protein with high affinity and specificity (26). SP-A also stimulates chemotaxis of alveolar macrophages (42). It is interesting, however, that collagen that can compete for high-affinity binding does not stimulate oxygen radical release, as SP-A does, nor stimulate macrophage movement (42). Previous studies showed that unlabeled SP-A competed much more effectively for binding than did either collagen or Clq (26), which might be one explanation for our present findings. It is also possible that SP-A interacts with macrophages through more than one mechanism and that part of the binding may be mediated via the noncollagen-like domain of the protein. The half-maximum binding for SP-A to alveolar macrophages occurred at - 4 pg/ml, and the dissociation con- stant as determined from kinetic binding experiments was 3.2 pg/ml (26). This is very close to the ECsO for surface-coated SP-A releasing oxygen radicals, which we determined in the present study to be 1.15 pg/ml. Such finding strongly supports the view that the oxygen radical release induced by surface-coated SP-A is receptor mediated.

Macrophage activation by surface-coated SP-A could be modified by other substances that are present in the alveolar lining layer, especially by surfactant phospholipids. We did not attempt to analyze effects of other than SP-A purified surfactant components on alveolar macrophages, although we showed slight inhibition of the chemiluminescence response by whole surfactant. Haya- kawa et al. (14, 15) found natural surfactant or syn- thetic phospholipids inhibiting priming of macrophages in luminol-enhanced systems; Speer et al. (29) found surfactant phospholipids without effect on luminol- enhanced chemiluminescence induced with PMA or opsonized zymosan in peripheral monocytes; Webb and Jeska (39) analyzed luminol-dependent chemiluminescence of stimulated rat alveolar macrophages and found an increase when cells were preincubated in the lipid fraction of alveolar lining material, with oxidation of the unsaturated lipids responsible for this increase. A com- plete understanding of the various role(s) and interac- tive actions of pulmonary surfactant components in modulating host defenses in the alveolar space of the lung will clearly require further investigation.

Downregulation of inflammatory responses of alveolar cells is very important for the host, in order to maintain normal lung functions. Under certain conditions, however, inflammation could be beneficial, for instance when immunogens or irritants are inhaled. Such particles need to be eliminated quickly, and elimi- nation is promoted if particles are opsonized. We have used enhanced chemiluminescence as a measure for alveolar macrophage activation and found that SP-A could substitute for serum as an opsonin. The opsonic capacity of alveolar lining fluid is different to serum, since serum composition differs from alveolar lining fluid composition. In this regard, SP-A might serve as a substitute for Clq in the alveoli, because alveolar lavage fluids obtained from rabbits contain only trace amounts of Cl (8), and Clq was not detected in human bronchial lavage (28).

SP-A is a member of a family of proteins that are calcium-dependent lectins, sharing sequence homology in their carbohydrate-binding domains (5, 31). These lectins have highly variable functions, but some of them, such as serum mannose-binding protein, conglutinin, Clq, and SP-A containing collagen-like sequences, may be involved in modulating host defense mechanisms (11, 18, 23). It has been speculated that lectins may provide an early line of defense in circumstances in which antibody formation may be slower. It is easy to envision such a scenario in the lungs where inhaled immunogens and irritants meet with alveolar cells and actually have the opportunity to penetrate the epithelial/endothelial barrier. Thus it seems reasonable to speculate that there may exist in the alveolar lining fluid a first line of defense against infection that would act quickly before enough time had elapsed for antibodies to be formed. A good candidate for mediating such reactions in alveoli is SP-A, which specifically binds to alveolar macrophages with high affinity and modulates the release of reactive oxygen species from these cells. According to our results, one may consider the possibility of SP-A regulating the inflammatory response in the gas-exchange area of mammalian lungs.

This work was supported by Deutsche Forschungsgemeinschaft and Maria Sonnenfeld-Gedachnisstiftung.

This study was performed in partial fulfillment of MD Thesis requirements for S. Weissbach and A. Neuendank. Preliminary results have been reported previously (22).

Address for reprint requests: U. Pison, Dept. of Anesthesiology and Intensive Care Medicine, Universitatsklinikum Rudolf Virchow, Freie Universitat Berlin, Augustenburger Platz 1, 13344 Berlin, Germany.

Received 9 July 1993; accepted in final form 29 June 1994.

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Surfactant protein A modulates release of reactive oxygen species from alveolar macrophages