October Pathologiss Surfactant Protein D

American Journal of Pathology, Vol. 139, No. 4, October 1991Copyrigbt © American Association of Pathologiss

Surfactant Protein D

Increased Accumulation in Silica-induced PulmonaryLipoproteinosis

Edmond Crouch, Anders Persson, DonaldChang, and Deena ParghiFrom the Departments of Pathology and Medicine, JewvishHospital at Washington University Medical Center,St. Louis, Missouri

Surfactant protein D (SP-D) (CP4) is a collagenoussurfactant-associated carbobydrate binding proteinthat is synthesized and secreted by alveolar epithelialcell& Previous studies have shown that intratrachealadministration of crystalline silica to rats elicits a

marked increase in the alveolar accumulation ofsurfactantlipids and surfactantproteinA (SP-A). Theauthors examined the accumulation of SP-D usingthis animal model ofalveolar proteinosis. Immuno-peroxidase localization of SP-D studies at 2 weeksafter silica instillation showed intense staining ofintra-alveolar exudates, and cytoplasmic staining ofhypertrophic type II cells. Immunoelectron micros-copy showed that airspace SP-D was specifically as-sociated with granular materia4 but not tubular my-elin or other membranous structures. SP-D wasquantified in bronchoalveolar lavage by immunoas-say using antibodies specificforSP-D, and by reverse-phase HPLC after affinity purification of SP-D onmaltosyl-agarose. Within 2 weeks after silica instil-lation, there was a > 45-fold increase in lavage SP-Dper lung compared with saline controls; includingan almost ten-fold increase in the insoluble or sur-factant-associated protein. These studies indicatethat the extracellular accumulation ofSP-D is mark-edly increased in silica-induced lipoproteinosis, andthat SP-D is associated with amorphous componentsidentified by electron microscopy. (AmJPathol 1991,139:765-776)

The epithelial surfaces of the lungs are covered by a thinfilm of a biochemically heterogeneous and lipid-rich ma-terial referred to as pulmonary surfactant. Surfactantplays important roles in regulating the surface active and

permeability properties of the alveolar wall, and couldalso contribute to the defense of the lung against micro-organisms.

In certain pathologic states, there is a marked accu-mulation of surfactant-rich lipoproteinaceous exudatewithin the alveoli, referred to as alveolar proteinosis. Hu-man pulmonary alveolar proteinosis (PAP) is usually idio-pathic; however, histologically similar reactions can beobserved in immunosuppressed patients, particularly inthe setting of hematologic malignancy.1" Lipoproteino-sis can also result as a consequence of massive inhala-tion of particulate materials including crystalline silica oraluminum.4- Regardless of the cause, the accumula-tions of airspace exudate interferes with normal gas ex-change, and with macrophage-mediated alveolar clear-ance.79

Ultrastructurally, the abnormal airspace material con-sists of tubular myelin-like structures, membranous vesi-cles, amorphous granular material, and rare lamellarbodies.1013 Biochemical and immunologic analyses ofinsoluble material from lavage of patients with alveolarproteinosis have shown surfactant phospholipids andproteins, specifically surfactant protein A (SP-A).14,15,3

The pathogenesis of human alveolar proteinosis, andthe contributions of abnormal surfactant synthesis andturnover, are still poorly understood.9 However, the de-velopment of silica-induced lipoproteinosis in rats is as-sociated with increased biosynthesis and secretion ofsurfactant lipids and SP-A.1r- 20We have identified a novel collagenous glycoprotein,

designated surfactant protein D (SP-D).21-24 The protein,which is synthesized by type 11 pneumocytes, can beisolated both as a soluble protein, and in association withinsoluble components of rat, bovine, and human bron-choalveolar lavage (BAL) (22.23 and unpublished data).Functional and structural studies indicate that SP-D is acalcium-dependent carbohydrate-binding protein, that issimilar in domain structure to conglutinin, SP-A, and the

Supported by Program Project Grant HL-29594 and NIH Grant HL-44015.Accepted for publication June 3, 1991.Address reprint requests to Dr. Edmond C. Crouch, Department of

Pathology, Jewish Hospital, 216 S. Kingshighway, St. Louis, MO 63110.

765

766 Crouch et alAJP October 1991, Vol. 139, No. 4

circulating mannose-binding proteins.24 In these studies,the accumulation of SP-D using the rat model of silica-induced alveolar proteinosis is examined.

Materials and Methods

Induction of LipoproteinosisSprague-Dawley rats were obtained from Charles River,and anesthetized by intraperitoneal injection of sodiumpentobarbital. Silica (Min-U-Sil, 5 ,um; U.S. Silica, BerkleySprings, WV) was washed and sterilized as described byDethloff et al.,16 and administered intratracheally underdirect visualization at a dose of 40 mg/rat in 0.4 ml ofsterile saline. After the desired interval, rats were re-anesthetized and sacrificed by exsanguination. Broncho-alveolar lavage (BAL) fluid was collected by repeatedlavage to a minimum total volume of 50 ml per rat.2223Cells were removed by centrifugation for 10 minutes at200 x g, and the crude surfactant pellet was collected bycentrifugation at 10,000 x g for 30 minutes at 4°C. Forquantitative studies, no additional washing of the pelletwas performed.

Purification and Quantification of SP-D byReverse-phase HPLCSP-D in the 10,000 x g supernatant was isolated by af-finity chromatography on maltosyl-agarose. SP-D asso-ciated with crude surfactant was extracted with 10 mMEDTA before affinity chromatography in the presence ofexcess calcium.24 SP-D was resolved from minor con-taminants by HPLC using a C4 reverse-phase columnequilibrated with 30% acetonitrile-0.1% trifluoroaceticacid (TFA) and eluted with a linear gradient of 30 to 70%acetonitrile in 0.1% TFA.' The eluted protein was quan-tified by absorbance at 214 nm using the integrated peakarea, and an extinction coefficient derived by amino-acidanalysis of the HPLC-purified protein. The assay was re-producible and shown to be linear in the range of 0.1 to30 ,ug SP-D and with injection volumes as high as 1 ml(Figure 6A). The cumulative recovery after maltosyl aga-rose and HPLC chromatography was approximately65% based on the recovery of exogenous radioiodinatedSP-D. Amino-acid analysis was performed as previouslydescribed.23

SDS-PAGEProteins were resolved by SDS-PAGE on 5%/10% dis-continuous methylenebis(acrylamide) slab gels and sil-ver stained.22 Gels included globular protein standards.Proteins were quantified by dye binding assay using BSAas standard.25 Immunoblotting was performed as previ-ously described.23

Antibody Preparation and Characterization

Polyclonal antisera to SP-D were prepared in rabbits, pu-rified, and characterized as previously described.23 Tofurther ensure specificity for ultrastructural studies, anti-bodies were purified by affinity chromatography on ratSP-D agarose. Approximately 0.5 mg of A-15M purifiedSP-D was coupled to 4 ml of Affi-Gel 10 (Bio-Rad, Rich-mond, CA) according to the manufacturer's instructions.IgG derived from 1 ml of whole antiserum was isolated bychromatography on a Zeta-Chrom-QAE disk (AMF Lab-oratory Products, Meriden, CT). The IgG was applied tothe affinity column at 4°C in phosphate-buffered saline(PBS), and bound antibodies were eluted with 0.2 M gly-cine, pH 2.3. Peak fractions were immediately neutralizedwith Tris base, pooled, and concentrated by ultrafiltrationusing a YM-10 membrane (Amicon, Beverley, MA).Greater than 90% of the immunoreactivity was boundand eluted from the column as assessed by ELISA. Theaffinity-purified antibody was stored at 4°C (or in smallaliquots at - 80°C) in the presence of 1 mg/ml BSA and0.02% (w/v) sodium azide. Dot-blot assays were per-formed as previously described.24

Histology and Immunoperoxidase Staining

Rat lung tissues were fixed by immediate immersion in4% neutral buffered formaldehyde and embedded inparaffin. For immunohistochemistry, 5 jirm sections werefixed to glass slides with 1% (v/v) Elmer's glue, deparaf-finized, and rehydrated. Endogenous peroxidase wasblocked by treatment with 0.03% (v/v) H202 in methanolfor 20 minutes at room temperature. Sections were thenincubated for 20 minutes at room temperature with 0.1 %(w/v) trypsin (Sigma, Type 11, porcine pancreas) in PBS,pH 7.4, to "unmask" antigenic determinants. Nonspecificimmunoglobulin binding sites were blocked with 3% (v/v)normal goat serum. Sections were then washed, and in-cubated in a moist chamber for 1 hour at 37°C with affin-ity-purified antibody to SP-D or IgG (10 jig/ml) dilutedwith PBS in the presence of 1 mg/ml BSA. Negative con-trols consisted of non-immune rabbit IgG. Washed sec-tions were incubated for 20 minutes with affinity purifiedbiotin-conjugated goat antibody to rabbit IgG (BRL;1:1600 in PBS containing 1% (w/v) BSA), washed, andincubated for 20 minutes with a 1:400 dilution of strepta-vidin-horseradish peroxidase (BRL). Complexes were vi-sualized by incubation for 20 minutes with 3,3'-diaminobenzidine (Sigma; 0.5 mg/ml in 50 mM Tris-HCI,pH 7.4) and H202. For some experiments staining wasenhanced with nickel.26 Sections were washed, dehy-drated, mounted in Permount, and examined by light mi-croscopy.

Surfactant Protein D 767AJP October 1991, Vol. 139, No. 4

Immunoelectron Microscopy

Rat lung tissues or isolated surfactant were fixed for 1hour at 40C in 0.5% (v/v) glutaraldehyde-3% (w/v)paraformaldehyde, in PBS, pH 7.4. Reactive aldehydeswere blocked with 0.1 M lysine, pH 7.4, for 30 minutes at40C. For most experiments the specimens were alsopostfixed in 0.5% (w/v) OS04 for 30 minutes at 4C. Afterwashing in 0.1 M cacodylate buffer, the samples weredehydrated through graded alcohols, embedded in Poly-Bed 812 (Polysciences), thin sectioned, and transferredto nickel grids. For some preliminary experiments, sam-ples were embedded in LR White (Electron MicroscopyScience, Fort Washington, PA) or Lowicryl (Electron Mi-croscopy Science). Nonspecific binding sites wereblocked with 3% (w/v) ovalbumin in TBS containing0.05% Tween-20, and by washing with 3% (v/v) normalgoat serum. Grids were incubated with the rabbit anti-body to rat SP-D or control rabbit immunoglobulin over-

night at 4°C in a moist chamber. Sections were sequen-tially washed with 1% (w/V) BSA and 3% goat serum; andthen incubated with affinity-purified biotinylated goat anti-rabbit immunoglobulin (1:1600) for 30 minutes at roomtemperature. After washing, sections were incubated for1 hour at room temperature with streptavidin-gold (E.Y.Labs, Inc; 15 nm particles, 1:75), washed in distilled wa-ter, and counterstained with 0.2% uranyl acetate and 4%lead citrate.

Results

Development of Lipoproteinosis

At 1 week after silica instillation, the lungs of treated ratsshowed an active peribronchiolar and alveolar inflamma-tory reaction characterized by airspace exudates con-taining mononuclear cells and neutrophils, and by smallinterstitial collections of macrophages. Large numbers of

Figure 1. Light micrographs of hematoxylinand eosin stained sections oflungfrom silica-treated rats at 2 weeks following silica instil-lation. Top, lung showing granulomas (G)with granular exudates in the surroundingalveoli (X31, original magnification). Bot-tom, lung showing alveoli (A) containinggranular airspace material (x125, originalmagnification). Selected hypertrophic-appearing type IIpneumocytes (T2) are iden-tified.

4'

Jr

f; i...

I.;.I::bsVw .'.

A

'.e


birefringent silica particles were also observed. At 2 to 3weeks after instillation, the lungs of silica-treated ratsshowed a 3- to 4-fold increase in wet weight relative tocontrols, and were grossly consolidated. Histologically,the lungs showed numerous confluent granulomas, andlarge accumulations of finely granular eosinophilic mate-rial within the alveoli (Figure 1) The reactions were mostmarked around small airways, and were accompaniedby prominent enlargement of type 11 pneumocytes. His-tologic studies performed after BAL showed a markedreduction of airspace staining with only small amounts ofgranular exudate distal to areas of intense granuloma-tous inflammation. Light microscopic examination of the10,000 x g pellet of BAL showed eosinophilic granulardebris comparable to that observed in the sections oflung (not shown). Transmission electron microscopy ofthe airspace material in situ, and of the 10,000 x g pelletof BAL, showed numerous multilamellated and tubularmyelinlike structures, moderate amounts of electron-dense amorphous and microgranular material, membra-

nous vesicles, and occasional secreted lamellar bodies(see below). Degenerating cells and silica particles werealso identified.

Immunohistochemical Localization of SP-D

To identify sites of SP-D accumulation and possible cel-lular sites of increased SP-D production, representativelung tissues from control and silica-treated rats were em-bedded in paraffin and immunostained using monospe-cific antibodies to rat SP-D. Lungs from the silica-treatedanimals showed intense staining of the granular airspacematerial (Figure 2). There was also strong cytoplasmicstaining of hypertrophic type 11 pneumocytes lining alveoliassociated with areas of alveolar exudate, particularly inthe vicinity of small airways; the staining intensity wasmuch stronger than for pneumocytes in control lung or inuninvolved areas of lung parenchyma (Figure 2A). Thecytoplasmic staining was diffuse, but appeared to spare

Figure 2. Immunolocalization of SP-D inlungs at 2 weeks after silica instillation. Sec-tions offormaldebyde-fixed and paraffin-embedded tissue were reacted w,ith affinity-purified antibodies to rat SP-D (A,C,D) orcontrol IgG (B). Sites of antibody bindingwere then visualized using a biotinylated sec-ondary antibody/streptavidin-peroxidase de-tection system (see Methods). A: Low-powerview of lung reacted with anti-SP-D showingstrong staining of peribronchiolar pneumo-cytes (x 625, original magnification). B:Lung reacted with control IgG (x 125, origi-nal magnification). C & D: Lung sections re-acted with anti-SP-D (x31 & x125, respec-tively). Selected granulomas (G), bronchioles(B), type II cells Cr2), and areas of alveolarexudate (A) are identified.


vacuoles corresponding to extracted lamellar bodies(Figure 2D). In addition, there was strong apical stainingof nonciliated bronchiolar (Clara) cells. We observed nosignificant staining of lung sections incubated with con-trol IgG or preabsorbed antibody (Figure 2B).

Ultrastructural Localization of SP-D

Immuno-electron microscopy with colloidal gold wasused to identify sites of antigen localization within air-space exudates and isolated crude surfactant. Antibod-ies to rat SP-D selectively reacted with amorphous gran-ular material both within airspaces (Figures 3A & 3B), andin isolated crude surfactant (Figures 3C & 3D). The la-beled aggregates showed considerable variation in sizeand electron density, and could not be morphologicallydifferentiated from larger numbers of unlabeled aggre-gates. Labeling was sometimes also observed in asso-

ciation with granular material associated with multilamel-lated structures in alveolar macrophages (not shown),and in crude surfactant from control rats (Figure 3E).There was no labeling of tubular myelin, multilamellatedtubular myelin-like structures, secreted lamellar bodies,membranous vesicles, or degenerating cells in the intactlung or isolated surfactant. There was also no significantlabeling of sections incubated with control immunoglob-ulins or with antibodies adsorbed by affinity chromatog-raphy on SP-D. Labeled extracellular aggregates werenot identified following pre-extraction of surfactant with 10mM EDTA.

Immunoelectron microscopy was also used to identifysites of SP-D localization within type 11 pneumocytes. Af-finity purified antibodies to rat SP-D demonstrated label-ing of the cytoplasm of pneumocytes in the lungs of sil-ica-treated rats, particularly in the vicinity of small vesic-ular structures subjacent to the plasma membrane. Goldwas also identified overlying short segments of rough en-

Figure 2 (continued)

ph.

J.

-Ad.l

B i!j.' . r


-BA. II| I

Figure 4. Immunogold localization ofSP-D in type IIpneumocytes. Lung tissuefrom silica-treated rats was processedfor immunoelectronmicroscopy as described in Methods except without post-fixation in osmium. Sections were reacted with affinity purified antibody to rat SP-Dor control IgG, and sites ofantibody binding were then visualized using a biotinylated secondary antibody/streptavidin-gold detection systemas in Figure 3. A: Type Hpneumocyte showing labeling ofc-toplas-m (x 16,000, original magnification); B, C: Immunogold labeling oftypeI pneumocytes (x25,000, original magnification). Note gold in the vicinit)y ofsmall vesicular structures beneath theplasma membrane, andin association with segments ofrough endoplasmic reticulum (arrows). Representative sites of extracted lamellar bodies (LB) are identified.

doplasmic reticulum (Figure 4). Although postfixation withosmium allowed partial preservation of lamellar bodies,labeling of other cytoplasmic structures was reduced,and no labeling of lamellar bodies was observed. Therewas only weak labeling of pneumocytes in control lungsand no staining of sections reacted with normal IgG.

Isolation of SP-D from Silica-treated Rats

SP-D isolated from the lavage of silica-treated rats wasbiochemically indistinguishable from SP-D obtained fromcontrol animals.22'24 The proteins comigrated on SDS-PAGE in the presence and absence of sulfhydryl reduc-ing agents, showed the same apparent molecular size by

gel filtration chromatography on A-15M in the presenceof EDTA, demonstrated identical retention times on re-

verse-phase HPLC, and reacted strongly with antibodiesto purified rat SP-D. Amino-acid analysis showed virtuallyidentical compositions (Table 1). Both proteins showedcalcium-dependent binding to maltosyl-agarose.24

Quantification of Lavage SP-D

Western and dot-blot assays of extracts of crude surfac-tant showed a > 10-fold increase in surfactant-associated SP-D (Figure 5). Western blots also confirmedthe specificity of the antibody to SP-D in crude surfactant(lanes 1, 4), and showed the efficient extraction of SP-Dby EDTA or glucose (lanes 7-10).

Figure 3. Immunogold localization of SP-D. Lung tissues or isolated crude surfactantfrom silica-treated and control rats were processedfor immunoelectron microscopy as described in the Methods. Sections were reacted with antibody to rat SP-D (A-C, E) or control IgG (D).Sites ofantibody binding were then visualized using a biotinylated secondary antibody/streptavidin-gold detection system. A & B: Airspacematerial in sections of lungfrom a silica-treated rat reacted with anti-SP-D (X41,500 and X35,000, original magnification respectively);C: Isolated crude surfactantfrom silica-treated rats reacted with anti-SP-D (X25,000); D: Surfactant reacted with control IgG (X25,000);E: Surfactantfrom control rat reacted with anti-SP-D (x59,000). Note lack ofspecific labeling of lamellated (L) or tubular myelin-like (T)structures.

A


Table 1. Amino Acid Contposition ofSP-D (Residues/1000)

Residue Control n = 3 Silica

Cys 14 15Hyp 32 22Asx 77 78Thr 35 31Ser 66 55Glx 142 149Pro 48 62Gly 212 214Ala 113 112Val 26 19Met 8 10lIe 19 18Leu 60 61Tyr 6 6Phe 20 29Hyl 20 21His 4 7Lys 49 41Arg 47 40Trp N.D. N.D.

Pro + Hyp 80 84Lys + Hyl 69 62

In separate experiments, we quantified the amount oflavage SP-D by reverse-phase HPLC after partial purifi-cation of the protein by affinity chromatography on mal-tosyl agarose (Figure 6). Silica-treated rats showed a >

45-fold increase in soluble SP-D (70 ± 9 vs. 1.5 ± 0.2,ug/rat; mean ± SD), and an 8-fold increase in EDTAextractable, surfactant-associated SP-D (1.03 ± 0.24 vs.

0.13 ± 0.01 ,ug/rat). SP-D was also recovered in smallamounts from the 200 x g cell pellet of lavage from silica-treated animals (approximately 1.9 ,ug/rat). No attemptwas made to quantify "intracellular" SP-D. Lung surfac-tant or lamellar body preparations isolated by the usualsucrose density gradient procedures do not contain SP-D; furthermore, sucrose can effectively compete effec-tively with other ligands for the binding of SP-D to malto-syl-BSA (unpublished data).

Discussion

These studies show that the extracellular accumulation ofSP-D is markedly increased in silica-treated animals.Light microscopic immunoperoxidase studies of paraffin-embedded lung tissue performed 2 weeks after admin-istration of silica showed uniform and intense reaction ofgranular airspace material with antibodies to SP-D. Fur-thermore, assays of BAL from silica-treated rats demon-strated a > 45-fold increase in total SP-D relative to con-trols. The magnitude of this increase is slightly greaterthan observed for SP-A at the same time interval after

silica administration. For example, Kawada et al.18 ob-served a 1 0-fold increase in SP-A with an almost 4-foldincrease in the ratio of SP-A to total protein at 14 days. A20 to 40-fold increase in lavage SP-A was reported byKuroki et al.27 In more recent studies, Miller and associ-ates"O observed an 8.7-fold increase in extracellular SP-A, with a 23-fold increase in the intracellular fraction at 2weeks.20 We are not aware of published quantitative dataregarding associated alterations in the hydrophobic pro-teins (SP-B and SP-C).

The mechanism(s) responsible for the increased ex-tracellular accumulation of SP-D are not known. However,the increase probably involves increased production ofSP-D by type 11 pneumocytes. Increases in lung surfac-tant phospholipid biosynthesis have been well de-scribed,28'19 and recent studies have shown increasedsynthesis of SP-A and surfactant phospholipids by iso-lated pneumocytes.18'2The predominant level of cellularregulation responsible for the increased production ofSP-A is controversial. Miller et al.20 observed parallel in-creases in the synthesis of SP-A and in the steady-statelevels of SP-A mRNA. By contrast, Kawada et al.18showed increased SP-A synthesis without a detectableincrease in message. Preliminary cell-free translation andNorthern hybridization studies indicate that there is a sev-eral-fold increase in translatable SP-D mRNA per lung at2 weeks after silica instillation (21, and unpublished data);however, additional biosynthetic and molecular studiesare needed to document increased production of SP-D inthis model. A contribution of nonciliated bronchiolar cellsto the SP-D accumulation cannot be excluded. Althoughtype 11 cells greatly outnumber stained bronchiolar cells,the latter cells show more intense cytoplasmic staining.

The intense cytoplasmic staining of hypertrophicpneumocytes with antibodies to SP-D probably reflectsincreased SP-D synthesis. The pattern of immunogoldlabeling suggests that cellular SP-D is associated withsecretory compartments such as rough endoplasmic re-ticulum and golgi, rather than storage or degradative or-ganelles. Increases in cytoplasmic staining have beenobserved to accompany increased production of otherconstitutively secreted proteins; for example, fibroblastactivation is associated with increased collagen produc-tion and an increase in cytoplasmic staining for typeprocollagen.-9

The intense immunoperoxidase staining of hyper-trophic pneumocytes is also consistent with the results ofstudies by other investigators, who identified hyper-trophic (type IIB), as well as normal appearing (type IIA),pneumocytes in silica-treated rats.18'20 The hypertrophiccell population consists of larger cells with more numer-ous lamellar bodies.3'31 These cells have have an in-creased capacity to synthesize surfactant lipids and SP-A,18'20 and also have an increased capacity to undergoDNA synthesis in vitro.32 We suggest that the predomi-


Figure 5. Western and dot-blot asays of sufactant-associated SP-D at 2 weeks. Lavage was separate{ypooledfor controland silica-treated rats. A: 7be crudesurfactant pellets were extracted with 10 mM EDTA or100 mM glucose; and aliquots ofsring material, e-tracts, and the post-extaction residues wre subectedto SDS-PAGE in the presence of DMT Becaue of thelarger amounts of SP-D in lavage from silica-treatedanimals, 10-fold larger volumefractions were loadedfor the control samples (ae., 0.08 rat equialets forcontrol rats and 0 008 equivalents for silica-treatedanimals). Proteis were tansfered to nitroelluloe,and SP-D was viisualized ng antbodies to rat SP-Dand an indirect biotinylated secondary antibody-streptavidinperoxidase detection system. Lane 1, unex-tracted control rat surfactant; lane 2, EDTA extract ofsufactant; lane 3, glucose extract; lane 4, surfactantfrom silica-treated rats; lane 5, EDTA extact; lane 64glucose extract; lanes 7 &8, post-extraction residuesforconrol rat sufactant; laes 9 & 10, pos-extractionresiduesfor sufactnfr-om silica-treated rats. B: SP-Dextracted from the stfactant of control and silica-treated rats uisa compared by dot bloting. atractwere seriallv diluted 1:1 (left to rIght) beginning witb0.04 and 0.004 equivalent ofsurfactantfrom control(C) and silica-treated (S) rats, respectively Rows 1 & 2,EDTA extracts; rows 3 & 4, 100 mM glucose extracts;rows 5 & 64 100 mM maltose etracts. After rrecangfor the 10-fold diference in sting dilution, dtere isan >10-fold increase in immunoreactive sufactant-associated SP-D at 2 ueeksfollouwng silica instiZlation.

nantly peribronchiolar distribution of immunoreactive hy-pertrophic cells (Figure 2A) reflects the route of deliveryand predominant site of silica-induced injury.

The immunogold studies indicate that SP-D is nonuni-formly distributed within the airspace exudate, and thatthe protein is predominantly, if not exclusively, associatedwith a fraction of amorphous granular material. The lattercomponents are similar in appearance to aggregatespreviously identified in human PAP. According to Gilmore

et al.10 amorphous materials account for approximately30% of the volume of insoluble material, and similar amor-phous materials have been identified in small amounts inlavage from normal animals and healthy humans (12, andunpublished data). Although the numbers of labeled ag-gregates were decreased after extraction of SP-D withEDTA (not shown), it is not yet clear whether the labeledaggregates are comprised of, or simply associated with,SP-D. The former possibility is consistent with our obser-

VOLUME INJECTED (il)

80

60

40

20

S P S PL I I I

SILICA CONTROLvation that a fraction of isolated SP-D can undergo self-aggregation and precipitation at physiologic calciumconcentrations in vitro (unpublished data). On the otherhand, incubation of the surfactant with purified bacterialcollagenase resulted in a decrease in labeling without a

detectable decrease in the number of aggregates (notshown). Meaningful morphometric analysis was pre-cluded by marked variation in the size and distribution ofthe aggregates (both in situ and in isolated surfactant),and by difficulties in morphologically distinguishing be-tween labeled and unlabeled aggregates.

Immunogold studies showed no specific labeling ofosmiophilic membranous structures including tubularmyelin, tubular myelinlike structures, vesicles, or se-

creted lamellar bodies. Comparable results were ob-served using several different fixation protocols, and with

samples embedded in Lowicryl or LR White. Although we

Figure 6. Recovery ofSP-Dfrom lavage ofcon-trol and silica-treated rats. Lavage was pooledfor 100 control and 12 silica-treated rats. The

10,000X g supenants and crude surfactantpellets were isolated, and the surfactant-associated SP-D in the pellet was extracted with10 mM EDTA Aliquots of the supernatant orrecalified surfactant extract corresponding to20 control or 2 silica-treated rats were sepa-rately adsorbed to maltosyl-agarose. SP-D was

eluted with 10 mM EDTA and quantified byreverse phase HPLC A: Integratedpeak area as

a function of injected SP-D from control andsilica-treated animals. B: Histogram showing re-

covery ofSP-Dfrom lavage supernatants (S) andpellets (P)for both control and silica-treated an-imals. Data are given as the mean + S.D. forfour to five independent determinations.

cannot exclude the possibility that membrane-associated SP-D or lipid-associated SP-D ligands were

extracted during processing, similar conditions of fixationand postfixation with osmium have permitted postem-bedding immunolocalization of SP-A to tubular myelinand lamellar bodies in plastic-embedded tissues.' Ourresults are consistent with the observation that SP-D isefficiently extracted from crude surfactant with EDTA or

competing sugars in the absence of detergents, andsupport our hypothesis that SP-D is functionally differentfrom SP-A, despite its striking structural similarities.2223These results also emphasize the biochemical and prob-able functional heterogeneity of airspace lining materials.

Previous studies of human PAP have identified collag-enous proteins and peptides in association with the in-soluble fraction of lavage.3'4 35The identification of SP-Aas a major component of PAP lavage, and the presence


ECM

C1J-

a:

0

CD

z

20(

10(

cn:w0L0(ICD

Al% A-% Al%

n)v


of a collagenous domain within SP-A have provided apartial explanation for these observations.14'36,15'37'38However, as noted by White et al.,37 there are differencesbetween the deduced amino-acid sequences of SP-Aand protein-sequencing studies of certain collagenouscomponents isolated from lavage, raising the possibilitythat PAP lavage contains more than one type of collag-enous protein. In this regard, preliminary studies indicatethat lavage from patients with alveolar proteinosis containlarge amounts of SP-D.

Acknowledgments

The authors thank Dr. Ida Kargi for assistance in developing theimmunogold detection methods and Janet North for excellentsecretarial assistance.

References

1. Claypool WD, Rogers RM, Matuschak GM: Update on theclinical diagnosis, management, and pathogenesis of pul-monary alveolar proteinosis (phospholipidosis). Chest 1984,4:550-558

2. Rosen SH, Castleman B, Liebow AA, Enzinger FM, HuntRTN: Pulmonary alveolar proteinosis. N Engl J Med 1958,258:1123-1142

3. Singh G, Katyal SL, Bedrossian CWM, Rogers RM: Pulmo-nary alveolar proteinosis. Staining for surfactant apoproteinin alveolar proteinosis and in conditions simulating it. Chest1983,1:82-86

4. Buechner HA, Ansari A: Acute silico-proteinosis: A newpathologic variant of acute silicosis in sandblasters charac-terized by histologic features resembling alveolar proteino-sis. Dis Chest 1969, 55:274-284

5. McEuen DD, Abraham JL: Particulate concentrations in pul-monary alveolar proteinosis. Environ Res 1978,17:334-339

6. Miller RR, Churg AM, Hutcheon M, Lam S: Pulmonary alve-olar proteinosis and aluminum dust exposure. Am RevRespir Dis 1984,130:312-315

7. Golde DW, Territo M, Finley TN, Cline MJ: Defective lungmacrophages in pulmonary alveolar proteinosis. Ann InternMed 1976, 85:304-309

8. Gonzalez-Rothi RJ, Harris JO: Pulmonary alveolar proteino-sis. Further evaluation of abnormal alveolar macrophages.Chest 1986, 5:656-661

9. Hoffman RM, Dauber JH, Rogers, RM: Improvement in al-veolar macrophage migration after therapeutic whole lunglavage in pulmonary alveolar proteinosis. Am Rev Respir Dis1989, 139:1030-1032

10. Gilmore LB, Talley FA, Hook GER: Classification and mor-phometric quantitation of insoluble materials from the lungsof patients with alveolar proteinosis. Am J Pathol 1988,133:252-264

11. Hook GER, Bell DY, Gilmore LB, Nadeau D, Reasor MJ,Talley FA: Composition of bronchoalveolar lavage effluents

from patients with pulmonary alveolar proteinosis. Lab In-vest 1978, 39:342-357

12. Hook GER, Gilmore LB, Talley FA: Dissolution and reassem-bly of tubular myelin-like multilamellated structures from thelungs of patients with pulmonary alveolar proteinosis. LabInvest 1986, 55:194-208

13. Takemura T, Fukuda Y, Harrison M, Ferrans VJ: Ultrastruc-tural, histochemical, and freeze-fracture evaluation of multi-lamellated structures in human pulmonary alveolar proteino-sis. Am J Anat 1987, 179:258-268

14. Crawford SW, Mecham RP, Sage H: Structural characteris-tics and intermolecular organization of human pulmonary-surfactant-associated proteins. Biochem J 1986, 240:107-114

15. Ross GF, Ohning BL, Tannenbaum D, Whitsett JA: Struc-tural relationships of the major glycoproteins from humanalveolar proteinosis surfactant. Biochim Biophys Acta 1987,911:294-305

16. Dethloff LA, Gilmore LB, Brody AR, Hook GER: Induction ofintra- and extra-cellular phospholipids in the lungs of ratsexposed to silica. Biochem J 1986, 233:111-118

17. Heppleston AG: Animal Model: Silica-induced pulmonaryalveolar lipo-proteinosis. Am J Pathol 1975, 78:171-174

18. Kawada H, Horiuchi T, Shannon JM, Kuroki Y, Voelker DR,Mason RJ: Alveolar type II cells, Surfactant Protein A (SP-A),and the phospholipid components of surfactant in acute sil-icosis in the rat. Am Rev Respir Dis 1989,140:460-470

19. Miller BE, Hook GER: Stimulation of surfactant phospholipidbiosynthesis in the lungs of rats treated with silica. BiochemJ 1988, 253:659-665

20. Miller BE, Bakewell WE, Katyal SL, Singh G, Hook GER:Induction of surfactant protein (SP-A) biosynthesis and SP-AmRNA in activated type 11 cells during acute silicosis in rats.Am J Respir Cell Mol Biol 1990, 3:217-226

21. Crouch EC, Rust K, Persson A, Mariencheck W, Moxley M,Longmore W: Primary translation products of pulmonarysurfactant protein D. Am J Physiol 1991, 260:L247-L253

22. Persson A, Rust K, Chang D, Moxley M, Longmore W,Crouch E: CP4: A pneumocyte-derived collagenous surfac-tant-associated protein. Evidence for heterogeneity of col-lagenous surfactant proteins. Biochemistry 1988, 27:8576-8584

23. Persson A, Chang D, Rust K, Moxley M, Longmore W,Crouch E: Purification and biochemical characterization ofCP4 (SP-D): a collagenous surfactant-associated protein.Biochem J 1989, 27:6361 -6367

24. Persson A., Chang D, Crouch E: Surfactant protein D (SP-D)is a divalent cation-dependent carbohydrate binding pro-tein. J Biol Chem 1990, 265:5755-5760

25. Rubin RW, Warren RW: Quantitation of microgram amountsof protein in SDS-mercaptoethanol-tris electrophoresis sam-ple buffer. Anal Biochem 1977, 83(2):773-777

26. Kuhn IlIl C, Boldt J, King Jr. TE, Crouch E, Vartio T, McDon-ald JA: An immunohistochemical study of architectural re-modeling and connective tissue synthesis in pulmonary fi-brosis. Am Rev Respir Dis 1989, 140:1693-1703

27. Kuroki Y, Mason RJ, Voelker DR: Pulmonary surfactant ap-oprotein a structure and modulation of surfactant secretion


by rat alveolar type 11 cells. J Biol Chem 1988, 263:3388-3394

28. Dethloff LA, Gladen BC, Gilmore LB, Hook GER: Kinetics ofpulmonary surfactant phosphatidycholine metabolism in thelungs of silica-treated rats. Toxicol Appl Pharmacol 1989,98:1-11

29. McDonald JA, Broekelmann TJ, Matheke ML, Crouch EC,Koo M, and Kuhn IlIl C: A monoclonal antibody to the car-boxyterminal domain of procollagen type visualizes colla-gen synthesizing fibroblasts. Detection of an altered fibro-blast phenotype in lungs of patients with pulmonary fibrosis.J Clin Invest 1986, 78:1237-1244

30. Miller BE, Dethloff LA, Hook GER: Silica-induced hypertro-phy of type 11 cells in the lungs of rats. Lab Invest 1986,55:153-163

31. Miller BE, Dethloff LA, Gladen BC, Hook GER: Progressionof type 11 cell hypertrophy and hyperplasia during silica-induced pulmonary inflammation. Lab Invest 1987, 57:546-554

32. Panos RJ, Suwabe A, Leslie CC, Mason RJ: Hypertrophicalveolar type II cells from silica-treated rats are committed toDNA synthesis in vitro. Am J Respir Cell Mol Biol 1990,3:51-59

33. Coalson JJ, Winter VT, Martin HM, King RJ: Colloidal goldimmunoultrastructural localization of rat surfactant. Am RevRespir Dis 1986, 133:230-237

34. Bhattacharyya SN: Characterization of collagenous andnon-collagenous peptides of a glycoprotein isolated fromalveoli of patients with alveolar proteinosis. Biochem J 1981,193:447-457

35. Bhattacharyya SN, Lynn WS: Structural studies on a colla-gen-like glycoprotein isolated from lung lavage of normalanimal. Biochim Biophys Acta 1980, 626:451-458

36. Phelps DS, Taeusch HW, Benson B, Hawgood S: An elec-trophoretic and immunochemical characterization of humansurfactant-associated proteins. Biochim Biophys Acta 1984,791:226-238

37. White RT, Damm D, Miller J, Spratt K, Schilling J, HawgoodS, Benson B, Cordell B: Isolation and characterization of thehuman pulmonary surfactant apoprotein gene. Nature 1985,317:361-363

38. Whitsett JA, Hull W, Ross G, Weaver T: Characteristics ofhuman surfactant-associated glycoproteins A. Pediatr Res1985, 19:501-508

Documents

October Pathologiss Surfactant Protein D