Ž .Journal of Immunological Methods 220 1998 139–149

A novel method of purifying lung surfactant proteins A and Dfrom the lung lavage of alveolar proteinosis patients and from

pooled amniotic fluid

Peter Strong a, Uday Kishore a, Cliff Morgan b, Andres Lopez Bernal c,Mamta Singh d, Kenneth B.M. Reid a,)

a Medical Research Council Immunochemistry Unit, Department of Biochemistry, UniÕersity of Oxford, South Parks Road, Oxford OX13QU, UK

b Department of Anaesthesia and IntensiÕe Care, Royal Brompton National Heart and Lung Hospital, London, SW3 6NP, UKc Nuffield Department of Obstetrics and Gynaecology, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK

d Centre for Biochemical Technology, Council for Scientific and Industrial Research, Mall Road, Delhi 110007, India

Received 15 December 1997; revised 3 July 1998; accepted 17 August 1998

Abstract

A simple procedure has been developed for the purification of the surfactant proteins SP-A and SP-D from lung lavage ofpatients with alveolar proteinosis. The SP-D is purified by fractionation of the supernatant obtained after spinning the lavageat 10 000=g for 40 min, while the bulk of the SP-A is purified by fractionation of the pellet. The supernatant is applied to amaltosyl–agarose column and the bound SP-D is specifically eluted using MnCl . The pellet is solubilised in 6 M urea and,2

following renaturation, the solubilised proteins are applied to maltosyl–agarose and SP-A eluted using a gradient of EDTA.Both SP-A and SP-D are further purified by gel-filtration on Superose-6. This procedure has also been used to preparesuccessfully human SP-A and SP-D from amniotic fluid and may be generally applicable to the isolation of these surfactantproteins from lung washings obtained from other species. q 1998 Elsevier Science B.V. All rights reserved.

Keywords: Surfactant proteins; Purification; Collectins; Carbohydrate recognition domains; Lung lavage; Amniotic fluid

1. Introduction

Ž . Ž .Lung surfactant proteins A SP-A and D SP-Dbelong to a family of collagen containing, C-type

Abbreviations: SP-A: human lung surfactant protein A; SP-D:human lung surfactant protein D; BAL: bronchoalveolar lavage;PC: phosphatidylcholine; DPCC: dipalmitoyl phosphatidylcholine;PI: phosphatidylinositol; EDTA: ethylene-diamino-tetraacetic acid

) Corresponding author. Tel.: q44-1865-275353; Fax: q44-1865-275729; E-mail: kbmreid@bioch.ox.ac.uk

lectins, called collectins. The primary structure ofeach of the collectins—mannose-binding lectinŽ . Ž .MBL , SP-A, SP-D, bovine conglutinin BC and

Ž .collectin-43 CL-43 —is organised into four regions:an amino terminal region involved in the formationof interchain disulphide bonds, a collagenous regioncomposed of Gly-Xaa-Yaa repeats, an a-helical neckpeptide, and a carboxy-terminal C-type carbohydrate

Ž . Ž .recognition domain CRD Holmskov et al., 1994 .The collectins are large oligomeric structures, eachassembled from multiple copies of a single polypep-

0022-1759r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved.Ž .PII: S0022-1759 98 00160-4

( )P. Strong et al.rJournal of Immunological Methods 220 1998 139–149140

tide chain, or two closely related polypeptide chainsin the case of SP-A. The collagenous regions of threepolypeptide chains assemble to form a collagen-liketriple helix, thus producing a subunit containing atriple-helical region, with three C-type lectin do-mains, at its C-terminal end. Six of these triple-heli-cal subunits make up the overall structure of SP-A,while SP-D is composed of a cruciform-like struc-ture, with four arms of equal length. SP-A binding,via its CRDs to the surface of pathogens, appears toenhance recognition of the microorganisms bymacrophages, leading to increased phagocytosis and

Žrespiratory burst activity by the phagocytic cells van.Iwaarden et al., 1990, 1991; Geertsma et al., 1994 .

Collectins play a role in innate immunity by recog-nising carbohydrate targets on pathogens, with, forexample, SP-A binding to carbohydrate structures onStreptococcus pneumoniae and Staphylococcus au-

Ž .reus McNeely and Coonrod, 1993 and SP-D bind-Ž .ing to lipopolysaccharides LPS of various Gram-

Ž .negative bacteria Kishore et al., 1996 . SP-A isconsidered to be important for the formation oftubular myelin and other surfactant aggregates, andfor the adsorption and spreading of surface activephospholipids at the air–liquid interface of alveoli. Italso augments lipid uptake by type II cells andinhibits the secretion of surfactant from type II cells,suggesting that it has a role in surfactant homeostasiswithin the alveolus. However, recent studies on ge-netically engineered SP-A deficient mice show thatthe animals have essentially normal lung surfactantfunction but show an increased susceptibility for

Žgroup B streptococcal infections Korfhagen et al.,.1996; LeVine et al., 1997 , thus supporting the view

that SP-A plays an important role in innate immunityin the lung. SP-A and SP-D have recently beenshown also to bind to various allergens, such as

Žthose derived from the house dust mite Wang et al.,. Ž1996 and the fungus Aspergillus fumigatus Madan

.et al., 1997a,b . Both the surfactant proteins havebeen shown to inhibit the ability of allergen-specificIgE, from patients suffering from dust mite allergy aswell as allergic bronchopulmonary aspergillosisŽ .ABPA , to bind respective glycoprotein allergens,thereby blocking allergen-induced histamine releasefrom the basophils of allergic patients. These obser-vations have led to the notion that SP-A and SP-Dcould be involved in the modulation of allergic

sensitisation andror development of allergic reac-tions. SP-A and SP-D also seem to have an impor-tant immunological role in the early antifungal de-fence responses in the lung, by inhibiting the infec-tivity of the spores through agglutination, and byenhancing uptake and killing of A. fumigatus spores

Ž .by phagocytic cells Madan et al., 1997a,b . Thisview is further supported by the finding that SP-Aand SP-D can both increase calcium-dependent neu-trophil uptake of Escherichia coli, S. pneumoniaeand Sta. aureus thus showing that surfactant col-lectins may promote neutrophil-mediated clearanceof bacteria in the lung independently of opsonising

Ž .antibody Hartshorn et al., 1998 .We report a simple and novel method of purifying

structurally and functionally intact SP-A and SP-Dmolecules using the same batch of lung lavage from

Žalveolar proteinosis patients. Large quantities up to.15 l of lung lavage fluid are collected from patients

undergoing treatment for Pulmonary Alveolar Pro-Ž .teinosis PAP . This is a very rare disease of un-

known aetiology resulting in the accumulation oflarge amounts of lipo-proteinaceous material withinthe alveolar space where it causes a progressivedeterioration in gas exchange. The only effectivetreatment is to physically wash out the material by

Ž .massive Whole Lung Lavage WLL . The techniqueof WLL has undergone gradual modification since it

Ž .was first described Ramirez et al., 1963 and is thesubject of occasional review in the medical literatureŽ .Claypool et al., 1985 . The purification procedureutilising the lavage fluid from the PAP patients,involves the use of MnCl to elute specifically SP-D2

bound to a maltosyl–agarose column, followed by anEDTA gradient to separate SP-A, bound to the mal-tosyl–agarose column, from other contaminatingproteins present in lung lavage, that also bind tomaltose. The SP-A is found mainly in the pelletobtained by spinning the lung lavage at 10 000=gfor 40 min and can be recovered by solubilisation in6 M urea, followed by renaturation and then use ofthe same affinity-chromatography and gel-filtrationprocedures used for SP-D. The method can also beused to purify SP-A and SP-D from amniotic fluid.The SP-A and SP-D preparations purified using thisprocedure were found to bind, in the expected man-ner, to carbohydrate–BSA conjugates and to phos-pholipids and lipopolysaccharides from Gram-nega-

( )P. Strong et al.rJournal of Immunological Methods 220 1998 139–149 141

tive bacteria. In addition, the SP-A, prepared utilis-ing urea, was shown to have the same functionalproperties as SP-A purified without the use of urea,as judged by their abilities to enhance the uptake andkilling of A. fumigatus, conidia by peripheral bloodmononuclear cells.

2. Methodology

2.1. Purification of SP-D using maltosyl–agarosechromatography

Ž .Human bronchoalveolar lavage BAL fluid wascollected from patients suffering from pulmonaryalveolar proteinosis, both lungs were lavaged fortherapeutic purposes, with a 1 week interval, using aheat exchanger and a modified perfusion apparatusto improve the efficiency of the lavage. Maltosyl–

Ž .agarose Sigma was packed into a FPLC columnŽ .10=30 mm and equilibrated with buffer contain-ing 20 mM Tris–HCl, pH 7.4, 10 mM CaCl , 0.02%2

Ž . Ž .wrv sodium azide TCB . The BAL, stored at 48C,was made 20 mM with respect to Tris–HCl, and 10mM with respect to EDTA, pH 7.4 and stirred for anhour at room temperature. The addition of EDTAhelps solubilise any aggregated SP-D. The turbid

Ž .solution 1 l after stirring was centrifuged at 10 000=g for 40 min at 48C to separate the SP-A richpellet from the supernatant which contains somesoluble SP-A and most of the SP-D. Prior to loading,the supernatant was made 20 mM with respect toCaCl and the pH readjusted to 7.4. The column was2

washed to background absorbance with TCB. Thiswas followed by washing with TCB containing 1 MNaCl to remove any non-specifically bound proteinssuch as albumin and a 9 kDa fragment of histone H4Ž .residues 24–134 that have been identified, by N-

Žterminal sequencing, to be present in BAL results.not shown . The SP-D, which has a specific require-

ment for calcium in binding to maltose, was elutedŽ .with 50 mM MnCl , 20 mM Tris–HCl, 0.02% wrv2

Ž .sodium azide, pH 7.4 Fig. 1 . The remaining bound

Fig. 1. NaCl wash and elution of SP-D with MnCl from the maltosyl–agarose column. Several proteins, including HSA and histone2

degradation products bind non-specifically to maltosyl–agarose and are readily eluted with high salt wash in the presence of calcium whileSP-D remains bound. SP-D is specifically eluted by replacing Ca2q with Mn2q when changing to elution buffer containing 50 mM MnCl2

after 25 ml of the sodium chloride high salt wash, as outlined in Section 2.

( )P. Strong et al.rJournal of Immunological Methods 220 1998 139–149142

Ž .proteins which includes SP-A were eluted with 100mM EDTA. The pooled fractions of SP-D weredialysed against 20 mM Tris–HCl, 100 mM NaCl, 5mM EDTA pH 7.4.

2.2. Extraction of SP-A from surfactant pellet

Surfactant pellet contains ;95% of the SP-A andis the best starting material for the purification of

Ž .SP-A. The BAL 1 l was made 20 mM with respectto Tris–HCl and 10 mM with respect to EDTA, pH7.4 and centrifuged at 10 000=g. The pellet wascollected and extracted with 20 mM Tris–HCl, 10mM EDTA, pH 7.4 containing 6 M Urea. Thisreadily solubilised the SP-A probably by disruptinghydrophobic interactions between SP-A molecules.A large volume of the Tris–HCl–EDTA–urea buffer

Ž .was used 100 mlrml pellet . The solution wasre-centrifuged at 10 000=g and any remaining pel-let re-solubilised with more Tris–HCl–EDTA–ureabuffer. The soluble fraction was dialysed against 10vol of 4 M, 2 M, 1 M urea in 20 mM Tris–HCl, 100

Ž .mM NaCl, 5 mM EDTA, pH 7.4 TEB and finallyinto TEB, over 12 h. The supernatant was made 15mM with respect to CaCl and loaded onto a malto-2

syl–agarose column, washed to background ab-sorbance with TCB and any SP-D eluted with MnCl2

Ž .as described above. The SP-A is eluted Fig. 2 withŽa linear EDTA gradient TCB to 100 mM EDTA, 20

mM Tris–HCl, pH 7.4 at a flow rate of 1 mlrmin.over 100 min . This leaches the divalent cations

Mn2q and Ca2q off the column and the variousproteins elute according to their relative bindingaffinities. Major contaminants were ferritin and IgGwhich eluted as a single peak at 10–15 mM EDTA,while SP-A eluted as a broad peak from 15–50 mMEDTA.

2.3. Gel filtration chromatography

The SP-A or SP-D pool was concentrated to 5 mlŽby ultrafiltration using an Amicon stirred-cell 10 000

.MW membrane and loaded onto a Superose 6Ž . Ž .preparative column 10 cm=30 cm Pharmacia

equilibrated with 20 mM Tris–HCl, 150 mM NaCl,5 mM EDTA, pH 7.4. SP-D, preparations purified bythe above method, elutes as a single peak with anapparent molecular weight of )1000 kDa. SP-Aelutes as a broad peak with an apparent molecularweight of 670 kDa. Low molecular weight contami-

Fig. 2. EDTA gradient elution of SP-A bound to the maltosyl–agarose column. The material prepared by resolubilising the 10 000=g pelletfrom BAL was applied to maltosyl–agarose. Following washing with 1 M NaCl and elution of any SP-D with MnCl , SP-A was eluted2

using an EDTA gradient. Impurities such as ferritin and IgG were eluted at approximately 10 mM EDTA. SP-A eluted between 15 and 50mM EDTA.

( )P. Strong et al.rJournal of Immunological Methods 220 1998 139–149 143

nants that co-purify with the proteins on the affinitycolumn are eluted late, close to the inclusion volumefor the column.

2.4. Isolation of SP-A and SP-D from amniotic fluid

Samples of human amniotic fluid were collectedvaginally at artificial rupture of the fetal membranes,carried out to induce or facilitate the progress oflabour, or in patients with spontaneous rupture of the

Žmembranes. All patients were at term G37 weeks.gestation and delivered healthy infants. This study

was approved by the Central Oxford Research EthicsCommittee. Amniotic fluid was pooled and storedfrozen at y208C. Prior to affinity chromatography,the amniotic fluid was spun at 10 000=g for 40 minat 48C after the addition of EDTA to 10 mM. Thesupernatant was mixed with 25 ml maltosyl–agaroseand calcium chloride added to 30 mM and the pHadjusted to 7.4. After mixing at 208C for 40 min, theresin was collected by low-speed centrifugation andwashed with TCB. After packing the resin in a 10cm=30 cm column, proteins were eluted by thesame procedure described in Section 2.1 for SP-D.SP-A was eluted with a gradient as described inSection 2.2.

2.5. N-terminal analysis

To confirm the amino-terminal sequence of thepurified SP-A, and to examine other proteins elutedfrom the maltosyl–agarose column, Edman degrada-tion was carried out using an Applied Biosystems470 A protein sequencer and on-line Applied Biosys-tems 120 A analysis of the amino acid derivatives.

2.6. Biotinylation of SP-A and SP-D

Purified SP-A and SP-D were biotinylated byincubating 0.6 ml of a 1 mgrml solution in 0.1 MNaHCO with 34.2 ml of 10 mM N-hydroxyl suc-3

Ž . Ž .cinimidobiotin BNHS Pierce Rockford, IL for 4 hat room temperature. Unreacted sulfo-NHS-biotinwas removed by dialysis against 10 mM sodiumphosphate, 150 mM NaCl pH 7.4 and any insolublematerial was removed by centrifugation in a mi-crofuge before use.

2.7. Phospholipid and carbohydrate binding assays

The phospholipids dipalmitoyl phosphatidyl-Ž . Ž .choline DPCC , phosphatidylinositol PI , and phos-

Ž . Ž .phatidylcholine PC all obtained from Sigma , wereŽ . Ždissolved in ethanol 10 mgrml , and samples 100

.mlrwell of each solution were applied separately tomicrotitre plates and air dried. The BSA conjugates

Ž .of maltose, mannose and glucose Sigma , wereŽ .dissolved in PBS 10 mgrml , and 100 mlrwell was

applied separately to the microtitre plates and al-lowed to bind overnight at 48C. The wells were

Ž .incubated with Tris–HCl–saline buffer 300 ml con-Ž . Ž .taining 2% wrv BSA and 10 mM CaCl TSCB2

for 1 h at 378C to block non-specific binding. Bi-Žotinylated SP-A or SP-D zero to 20 mgrml in

.TSCB were added to the wells and incubated for 2 hat 378C. After the microtitre plates had been washedfive times with TSCB buffer, ExrAvidin-alkalinephosphatase conjugate 1:5000 dilution in TSCB, wasadded to each well. After the pellet had been washed

Ž .with Tris buffer, p-nitrophenylphosphate pNPP wasadded as a substrate for the phosphatase reaction,and the plates were incubated for 30 min at 378C.The reaction was stopped by the addition of 1 MNaOH and the absorbance was read at 405 nm.

2.8. Killing actiÕity of A. fumigatus conidia by pe-( )ripheral blood mononuclear cells PBMCs in the

presence of SP-A

Human PBMCs were isolated from heparinisedblood which was layered on to an equal volume ofFicoll hypaque and centrifuged at 1500=g for 7min. The buffy coat, rich in PBMCs, was carefullyaspirated, washed with RPMI 1640 and suspended in

Ž .RPMI 1640 containing 10% vrv fetal calf serumand then plated on tissue culture plates. The cellpreparation was judged to be 95% pure. The purityand viability of the preparation was assessed bycounting the cells on a haemocytometer after stain-ing with trypan blue.

The antifungal activities of PBMCs in the pres-Žence of both forms of SP-A SP-A isolated from

.supernatant vs. urea-extracted were determined byŽ w xthe MTT 3- 4,5-dimethylthiazol-2-yl -2,5-dephenyl-

. Žtetrazolium bromide colorimetric assay Levitz and

( )P. Strong et al.rJournal of Immunological Methods 220 1998 139–149144

.Diamond, 1985 . The viable cells take up MTT andoxidise it to tetrazone in their mitochondria, whereasdead cells are not capable of oxidising MTT. PBMCsŽ 6 . Ž .10 rml 100 mlrwell in RPMI medium with 5%Ž .wrv fetal calf serum were plated onto 96-well

Ž .tissue culture plates Nunc and were allowed toŽ .adhere in a humidified 5% vrv CO incubator at2

378C. The supernatant medium was aspirated care-fully without disturbing the monolayer. A total of

6 Ž100 ml of 10 conidiarml prepared in PBS contain-2q 2q. Žing Mg and Ca , in RPMI medium without

.serum containing various concentration of treatedand untreated SP-A were added to each well, and theplates were incubated at 378C for 1 h. The super-natants were discarded, and 100 ml of 0.5% deoxy-cholic acid per well in sterile PBS was added to lysethe macrophages. The plates were then washed threetimes with sterile water. Subsequently, 0.5 mg ofMTT per ml in RPMI medium was added to eachwell, and the plates incubated at 378C for 3 h. Thewells were then aspirated dry, and 200 ml of iso-propanol was used to extract dye from each well,volumes of 150 ml were transferred to another 96-well microtitre plate, and the absorbance was mea-sured at 562 nm. PBMCs alone and conidia alone

were taken as negative and positive controls, respec-tively.

3. Results

3.1. Preparation of SP-D

The supernatant, obtained by spinning BAL froman alveolar proteinosis patient at 10 000=g for 40min, was applied to a maltosyl–agarose column and,after washing with buffer containing 1 M NaCl, theSP-D was eluted with buffer containing 50 mM

Ž .MnCl Fig. 1 . The SP-D pool from the maltosyl–2

agarose column ran as a single peak with an apparentmolecular weight of greater than 1000 kDa on aSuperose 6 gel-filtration column. The expectedmolecular weight of the unaggregated tetrameric formof human SP-D, which is composed of 12 identicalpolypeptide chains of 43 kDa, is 516 kDa and there-fore, this method of preparation gives an aggregatedform of SP-D which is much larger than, for exam-

Ž .ple, the 670 kDa SP-A molecule Fig. 3 . In Fig. 3the behaviour of a mixture of SP-D and SP-A prepa-rations, from maltosyl–agarose, is shown on gel

Fig. 3. Gel filtration of a mixture of analysis of SP-A and SP-D preparations on Superose 6. A mixture of SP-A and SP-D is shown toŽ .illustrate that the SP-D is eluted as a high molecular weight aggregate )1000 kDa rather than the expected 546 kDa molecular weight for

Ž .SP-D, whereas the apparent molecular weight of SP-A 670 kDa is close to its expected molecular weight of 516 kDa. The gel-filtrationallows separation of SP-D and SP-A from low molecular weight contaminants.

( )P. Strong et al.rJournal of Immunological Methods 220 1998 139–149 145

filtration on Superose-6 for comparison purposes;this also illustrates the removal of some low molecu-lar weight contaminants. This procedure yields ap-proximately 100 mg SP-D per l of alveolar pro-teinosis-BAL, although yields do depend upon theseverity of the proteinosis. When the procedure wasused to prepare SP-D from pooled amniotic fluid ayield of approximately 15 mg SP-D per l of fluid wasobtained.

In most of the previous literature describing meth-ods of purification of SP-D and SP-A the yields arenot given; therefore, it is not possible to make com-parisons in terms of yields. One exception is thedescription of the purification of SP-D from amniotic

Žfluid where a yield of 50 mgrl was reported Lu et.al., 1992 .

3.2. Preparation of SP-A

ŽA small amount of SP-A 10–50 mgrl of BAL,.depending upon the severity of the disease can be

obtained from the supernatant, obtained by spinningBAL from an alveolar proteinosis patient at 10 000=g for 40 min, and fractionation as outlined for theSP-D preparation but with the final elution beingmade with 50 mM EDTA. Much larger amounts ofSP-A could be isolated by solubilising the pellet,obtained by spinning the BAL at 10 000=g in 6 Murea, followed by renaturation, affinity chromatogra-phy and gel-filtration. The SP-A was eluted as abroad peak from the maltosyl–agarose affinity col-umn, by 10–50 mM EDTA, immediately after a

Ž .peak containing IgG and ferritin Fig. 2 . Removal oflow molecular weight contaminants was achieved onSuperose 6 where the SP-A was eluted as a singlepeak with an apparent molecular weight of approxi-

Ž .mately 670 kDa Fig. 3 . The yield of purified SP-Awas 100–200 mgrml of the pellet obtained by spin-ning the BAL at 10 000=g for 40 min. The amountof pellet varied with the severity of alveolar pro-teinosis, from 0.5–1.0 ml pelletrl of BAL in mildcases to 5.0–10.0 ml pelletrl of BAL in severecases. In the case of amniotic fluid, the yield ofSP-A from the supernatant obtained by spinning at10 000=g for 40 min, was approximately 15 mgrl.No significant amount of SP-A could be recoveredfrom the pellet obtained by spinning the amnioticfluid at 10 000=g for 40 min.

3.3. SDS-PAGE analysis of purified fractions

Under reducing conditions SP-D gave a singlemajor band with an apparent molecular weight of 43

Ž .kDa on SDS-PAGE Fig. 4, lane 1 along withminor, higher molecular weight bands which ap-peared to be dimers and trimers of the 43 kDa band

Ž .as judged by immunoblotting results not shown .The SP-A gave two major bands under reducingconditions which corresponded to a polypeptide chainwith an apparent molecular weight of 32 kDa and a

Ž .64 kDa dimer of this chain Fig. 4, lane 2 . Undernon-reducing conditions the SP-D gave a single band

Ž .of apparent molecular weight of 145 kDa Fig. 5while the SP-A gave disulphide linked subunits ofgreater that 250 kDa which did not enter the 5%Ž . Ž .wrv SDS-PAGE gel Fig. 5 . There is no evidencefor contamination of the SP-A by SP-D or by lowmolecular weight proteins, such as HSA, as judgedby the non-reduced SDS-PAGE results.

Ž .Fig. 4. SDS-PAGE 10% wrv analysis of purified preparationsof SP-D and SP-A, under reducing conditions. Lane 1: SP-D. Themajority of SP-D is composed of a 43 kDa polypeptide chain,with faint bands corresponding to dimers and trimers of the 43

Ž .kDa chain confirmed by immunoblotting . Lane 2: SP-A. Twobands are seen, a major band corresponding to the 32 kDapolypeptide chain of SP-A plus a proportion of non-reducible

Ž .dimeric SP-A 64 kDa . Traces of higher oligomers and someŽ .aggregates confirmed by immunoblotting were also seen.

( )P. Strong et al.rJournal of Immunological Methods 220 1998 139–149146

Ž .Fig. 5. SDS-PAGE 5% wrv analysis of purified preparations of SP-A and SP-D under non-reducing conditions. Lane 1: Molecular weightŽ .markers kDa . Lane 2: SP-D which migrates as a single band of 145 kDa. Lane 3. SP-A which is characterised by disulphide-linked

structures larger than 250 kDa.

3.4. Analysis of the binding and functional propertiesof SP-A and SP-D

Since the isolation of SP-A from the surfactantpellet obtained by centrifugation of the lung lavageat 10 000=g involves the use of 6 M urea, acomparison was made between the binding abilitiesof SP-A prepared with, and without, the use of urea.It was found that the urea-treated SP-A had identicalbinding properties to the non-urea-treated SP-A with

Žrespect to binding to a number of carbohydrate BSA.conjugates of maltose, mannose and glucose and

Ž .phospholipid DPCC, PI and PC targets as judged

Žby the assays described in Section 2 results not.shown . Further evidence that urea treatment does

not damage SP-A was obtained when both untreatedand urea-treated SP-A were assayed in the MTTbased ‘killing’ assay of A. fumigatus conidia byPBMCs. It was found that both types of preparations

Ž .of SP-A gave the same values in this assay Table 1 .The binding and agglutination properties of the

SP-D isolated by this procedure were examined us-ing the E. coli agglutination assay described by

Ž .Kuan et al. 1994 . The SP-D prepared, using 50mM MnCl for elution from the maltosyl–agarose2

affinity column, showed the expected degree of ag-

Table 1Enhancement by SP-A of the uptake and killing of A. fumigatus conidia by PBMCs

Percentage of conidia killed

PBMCs alone PBMCsq PBMCsqnon-urea-treated SP-A urea-treated SP-A

Patient 1 58 84 97Patient 2 48 90 77Patient 3 51 75 77

( )P. Strong et al.rJournal of Immunological Methods 220 1998 139–149 147

glutination of E. coli in comparison with SP-Dpreparations eluted with EDTA or maltose from

Ž .affinity columns results not shown . This indicatesthat the use of manganese in the preparation of SP-Ddoes not have a deleterious effect on its biologicalactivity.

4. Discussion

Affinity chromatography using maltosyl–agaroseŽis frequently used to purify SP-D Persson et al.,

.1990; Lu et al., 1993 . Typically, the lung lavage iscentrifuged at 10 000=g which sediments much ofthe SP-A into the surfactant pellet. The SP-D in thesupernatant is bound to the maltosyl–agrose andeluted with EDTA. This method, by itself, leads toonly partially pure SP-D and is insufficiently selec-tive to separate SP-D from residual SP-A which alsobinds to maltosyl–agarose and elutes with EDTA.SP-A is usually extracted from the pellet, obtainedby high speed centrifugation of lung lavage. Thepellet is washed in calcium containing buffer toremove albumin and most of the SP-D. SP-A can bereleased from the surfactant pellet using EDTA or

Ž .EGTA Suwabe et al., 1996 . This method givesonly partial purification and is unable to separateSP-A from residual SP-D, IgG and ferritin. Addition-ally, a major proportion of SP-A remains associatedwith the surfactant pellet. The method described herepurifies SP-A and SP-D from the same batch of lunglavage or amniotic fluid and relies on selective elu-tion of SP-D by use of MnCl , thus rendering it free2

from SP-A. The pellet-associated SP-A in the alveo-lar proteinosis samples can be dissociated by 6 Murea and subsequent dialysis to remove urea yieldedfunctionally intact SP-A.

Our procedure is novel in three ways: firstly,Mn2q is used to specifically elute SP-D free fromSP-A; secondly, the use of an EDTA gradient toseparate SP-A from other contaminants that are boundto the maltosyl–agarose column, such as histonefragments, HSA, etc.; and thirdly, the use of 6 Murea to solubilise aggregated SP-A. This methodyields human SP-A and SP-D of high purity fromboth lung lavage and amniotic fluid and may begenerally applicable to the isolation of these surfac-tant proteins from lung lavages obtained from other

species. The carbohydrate-binding step on the malto-syl–agarose column selects only functionally intactSP-D and SP-A and it has been found that use of amannosyl–agarose column is as effective as the mal-tosyl–agarose in the purification of both SP-A andSP-D.

Previous studies have already examined the diva-lent cation requirements for SP-D binding to malto-syl–BSA and found that binding was inhibited by 2mM solutions containing barium, strontium, magne-

Ž .sium or manganese Persson et al., 1990 . However,an earlier study had found that strontium, barium andmanganese could substitute for calcium in mediatingthe binding of SP-A to immobilised mannoseŽ .Haagsman et al., 1997 . Both these sets of resultsare compatible with what is reported in this paper,i.e., the apparent inhibition of the binding of SP-D tocarbohydrate targets in the presence of manganeseand the retention of binding of SP-A to a carbo-hydrate in the presence of manganese.

The preparations of SP-A and SP-D did not ap-pear to be contaminated with IgG, IgM, and IgEimmunoglobulins as judged by ELISA using anti-hu-man IgG, anti-human IgM, and anti-human IgE per-oxidase conjugates. SP-A and SP-D, preparationspurified by the method described in this paper, havebeen used in various functional assays in our labora-tory. Although SP-A could be purified along withSP-D, using the soluble fraction of the lavage, it wasobtained in a relatively low yield compared to theamount of SP-A which could be recovered from theurea-extract of the surfactant pellet from the lavage.The SP-D, prepared by this method bound to BSAconjugates of maltose, mannose and glucose in theorder: maltose)mannose)glucose. SP-A boundstrongly to mannosyl–BSA, and showed little affin-ity for maltose or glucose conjugates. SP-D boundstrongly to PI and exhibited little binding to PC orDPCC while SP-A bound strongly to all three phos-

Ž .pholipids results not shown .In all the experiments, involving carbohydrate and

phospholipid binding, the urea-treated SP-A behavedidentically to the SP-A isolated without the use ofurea. Further confirmation that the urea treatmentdoes not damage SP-A was obtained when bothurea-treated, and untreated, SP-A behave in the samemanner in the MTT based ‘killing’ assay of A.fumigatus conidia by PBMCs. The SP-D, purified by

( )P. Strong et al.rJournal of Immunological Methods 220 1998 139–149148

Ž .the procedure using MnCl , has been found to: i2

enhance killing and phagocytosis of conidia of A.fumigatus by neutrophils and alveolar macrophagesŽ . Ž .Madan et al., 1997a,b ; ii generate superoxide

Žradicals from circulating neutrophils Madan et al.,. Ž . Ž1997a,b ; iii act as a chemoattractant Madan et al.,. Ž .1997a,b ; iv bind glycoprotein allergens of house

Ždust mite and inhibit IgE binding to allergens Wang. Ž .et al., 1996 and v block histamine release from

sensitised basophils when challenged with aller-gensrantigens of A. fumigatus, an opportunistic fun-gal pathogen that causes ABPA and invasive as-

Ž .pergillosis Madan et al., 1997a,b . A further indica-tion that MnCl does not have a deleterious effect on2

SP-D’s biological activity is that it shows the ex-pected degree of agglutination of E. coli in an assay

Ž .procedure described previously Kuan et al., 1994 .In conclusion, this method effectively purifies

functionally active forms of SP-A and SP-D thusproviding material for future research on the func-tions of these proteins.

Acknowledgements

We would like to thank Tony Willis for carryingout the N-terminal sequence analysis and AlisonMarsland for preparation of the manuscript. Thiswork was partly supported by the Medical Research

Ž .Council, UK PS, KBMR and the European Com-Ž .mission Biotechnology Programme UK, KBMR .

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