Purification, characterization and immunolocalization of porcine surfactant protein D

Authors


Dr O. L. Nielsen, Department of Veterinary Pathobiology, Laboratory of Pathology, The Royal Veterinary Agricultural University, Ridebanevej 3, 1870 Frederiksberg, Denmark.
Email: ole@kvl.dk

Summary

Surfactant protein D (SP-D) is a collectin believed to play an important role in innate immunity. SP-D is characterized by having a collagen-like domain and a carbohydrate recognition domain (CRD), which has a specific Ca2+-dependent specificity for saccharides and thus the ability to bind complex glycoconjugates on micro-organisms. This paper describes the tissue immunolocalization of porcine SP-D (pSP-D) in normal slaughter pigs using a monoclonal antibody raised against purified pSP-D. Porcine SP-D was purified from porcine bronchoalveolar lavage (BAL) by maltose-agarose and immunoglobulin M affinity chromatography. The purified protein appeared on sodium dodecyl sulphate–polyacrylamide gel electrophoresis as a band of ∼53 000 MW in the reduced state and ∼138 000 MW in the unreduced state. Porcine SP-D was sensitive to collagenase digestion and N-deglycosylation, which reduced the molecular mass to ∼24 000 MW and ∼48 000 MW respectively, in the reduced state. N-deglycosylation of the collagen-resistant fragment, reduced the molecular mass to ∼21 000 MW showing the presence of an N-glycosylation site located in the CRD. Porcine SP-D bound to solid-phase mannan in a dose and Ca2+-dependent manner with a saccharide specificity similar to rat and human SP-D. The purified protein was used for the production of a monoclonal anti-pSP-D antibody. The antibody reacted specifically with pSP-D in the reduced and unreduced state when analysed by Western blotting. Immunohistochemical evaluation of normal porcine tissues showed pSP-D immunoreactivity predominantly in Clara cells and serous cells of the bronchial submucosal glands, and to a lesser extent in alveolar type II cells, epithelial cells of the intestinal glands (crypts of Lieberkühn) in the duodenum, jejunum and ileum and serous cells of the dorsolateral lacrimal gland.

Abbreviations:
BAL

bronchoalveolar lavage

CRD

carbohydrate recognition domain

mAb

monoclonal antibody

pAb

polyclonal antibody

pSP-D

porcine surfactant protein D

SP-A

surfactant protein A

MBL

mannan-binding lectin

CL-43

collectin-43

CL-46

collectin-46

CL-L1

collectin-liver 1

CL-P1

collectin-placenta 1

Introduction

The pulmonary surfactant is a complex of lipids and proteins that coats the epithelial surface of the peripheral airspace and, among other things, serves to lower the surface tension at the air/fluid interface.1 Four lung surfactant proteins (SP) – SP-A, SP-B, SP-C and SP-D – have so far been identified. SP-A and SP-D belong to a family of hybrid proteins termed collectins. Collectins form oligomers of trimeric subunits, each composed of disulphide-bound monomers, containing a collagenous region and a C-terminal carbohydrate recognition domain (CRD).2 Eight different collectins have now been identified, including the pulmonary surfactant proteins SP-A3 and SP-D;4 mannan-binding lectin (MBL);5 the bovine collectins conglutinin6 collectin-43 000 MW (CL-43)7 and collectin-46 000 MW (CL-46);8 collectin-liver 1 (CL-L1);9 and collectin-placenta 1 (CL-P1).10 Collectins take part in innate immunity by binding complex glycoconjugates on a wide range of pathogenic micro-organisms with the presumed function of inhibiting infection, enhancing the clearance by phagocytes and modulating the immune response.11–15

Different biological functions have been ascribed to SP-D. For example, SP-D is thought to function antimicrobial by binding selectively to saccharides and lipids on microbial surfaces. The antimicrobial function is mediated by direct lysis, agglutination and/or neutralization of the micro-organisms; and subsequent opsonization for phagocytic cells.13,16 Studies with gene targeted mice also indicated an important role of SP-D in the regulation of surfactant lipid homeostasis and macrophage function.17 Furthermore, there are indications that SP-D binds to immature dendritic cells, stimulates the entry of pathogens into these cells and thereby might promote antigen presentation.18

Porcine respiratory diseases have major welfare and economic consequences and are today regarded as the most serious disease problem in modern swine production.19 A continuous search for proteins as potential new disease markers is taking place. Knowledge of pSP-D contributes to the understanding and development of porcine models for human respiratory infectious diseases.20 Furthermore, porcine lung surfactant has been used successfully as a therapeutic agent in different human surfactant disorders21–23 where the protein component of the surfactant is believed to play an important therapeutic role.24

The pSP-D cDNA sequence has previously been determined25 the protein described26 and the interaction between pSP-D and Influenza A virus described.27,28 Here we describe the purification and characterization of porcine SP-D, for the production and characterization of a specific monoclonal antibody (mAb) directed against porcine SP-D. This antibody was subsequently used for pSP-D immunolocalization in a wide range of tissues from normal slaughter pigs.

Materials and methods

Sampling and preparation of bronchoalveolar lavage (BAL)

Macroscopically normal lungs were collected from approximately 5-month-old slaughter pigs at an abattoir and subjected to BAL with a total of 7 l Tris-buffered saline (TBS: 10 mm Tris-base, 0·14 mm NaCl, 0·025% (v/v) HCl, 0·05% (w/v) NaN3, pH 7·4) containing 5 mm iodoacetamid (Sigma-Aldrich, Vallensbaek Strand, Denmark), 5 mm Cyclocapron® (Acidum tranexamicum; Pharmacia & Upjohn, Sweden), 10 units/ml Trasylol® (Aprotinin; Bayer, Leverkusen, Germany) as enzyme inhibitors and 5 mm ethylenediaminetetraacetic acid (EDTA). The BAL fluid was cleared by passage through gauze and centrifuged at 10 000 g for 30 min at 4° to separate the SP-A rich pellet from the SP-D rich supernatant. Finally, the supernatant was stored at 4°.

Maltose affinity chromatography

Porcine SP-D was purified by maltose agarose affinity on a computer monitored fast performance liquid chromatography system (FPLCdirector® Version 1.3; Pharmacia), using a modified version of a previously described method.29 Briefly the BAL fluid was adjusted to 15 mm CaCl2 pH 7·4, filtered trough a glass fibre filter and a membrane filter (0·45 µm, PALL Life Sciences, New York, NY), diluted twofold with TBS, 5 mm CaCl2, 0·05% (v/v) Emulphogene® (Polyoxyethylene 10 Tridecyl Ether; Sigma-Aldrich) and applied to a 15 ml maltose-agarose affinity column (Sigma-Aldrich). After washing away non-specifically bound proteins with TBS, 5 mm CaCl2, 1 m NaCl, 0·05% (v/v) Emulphogene® the collectin was eluted with TBS, 100 mm MnCl2, 0·05% (v/v) Emulphogene® using an initial MnCl2 step gradient of 10 ml TBS, 0·5 mm MnCl2, 0·05% (v/v) Emulphogene®. Selected fractions analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) were pooled and dialysed over night at 4° against TBS, 5 mm CaCl2, 0·05% (v/v) Emulphogene®.

Immunoglobulin M (IgM) affinity chromatography

The dialysed maltose affinity-purified protein-pool was diluted 2-fold with 20 mm NaH2PO4, 0·8 m (NH4)2SO4, pH 7·5 and loaded on a HiTrap IgM purification HP® column (Amersham Pharmacia Biotech, Hoersholm, Denmark). Washing and elution was performed in accordance with the directions given by the manufacturer. The lectin containing fractions from the flow through were analysed by SDS–PAGE and dialysed over night at 4° against TBS, 5 mm CaCl2, 0·05% (v/v) Emulphogene®.

SDS–PAGE and Western blotting

SDS–PAGE was performed in a 4% stacking gel and 20% separation gel with a discontinuous buffer system30 and the Mark 12TM molecular weight marker (Invitrogen, Taastrup, Denmark) as previously described.31 Protein bands were visualized by silver staining.32 In SDS–PAGE for Western blotting 4% stacking and 10% separation gels and the MagicMark molecular weight marker were used (InVitrogen).

Immunoblotting was carried out essentially as described previously33 in a blotting cell from Bio-Rad (Mini Trans Blot) using Immobilon P membranes (Millipore, Glostrup, Denmark). After transfer of protein from the gel (1 hr, 150 mA), the membranes were blocked for 10 min with TBS (5 mm Tris/HCl pH 7·2, 0·25 m NaCl) plus 2% Tween-20 (Merck). After washing in TBS plus 0·1% Tween-20 (washing buffer) the membranes were incubated overnight at 4° with 5 µg/ml mAb 1.7 anti pSP-D in washing buffer. After washing, incubation for 1 hr at room temperature was performed in 1/1000 alkaline phosphatase-coupled goat anti-mouse immunoglobulin (DAKOCytomation, Glostrup, Denmark) in washing buffer. Subsequently the blots were developed using NBT/BCIP tablets (Roche, Denmark) following the instructions of the manufacturer.

Collagenase digestion

Purified protein (1–10 µg) was incubated for 24 hr at 37° with 0·25 units of Clostridiopeptidase B (Clostridium histolyticum type VII; Sigma-Aldrich) in 25 mm Tris-HCL, 10 mm CaCl2, pH 7·4. Controls were prepared by adding 10 mm EDTA to the buffer. Digests and the control were analysed by SDS–PAGE.

N-deglycosylation

Purified, as well as collagenase-treated, SP-D was dissolved in 50 mm Na2HPO4, pH 8·0 and incubated for 24 hr at 37° with PNGase F® (N-glycosidase F from Flavobacterium meningosepticum; Boehringer Mannheim, Mannheim, Germany) at a concentration of 0·01 units/mol of protein. The preparations were analysed by SDS–PAGE.

N-terminal amino acid sequencing

The dialysed maltose and IgM affinity-purified protein-pool was subjected to N-terminal amino acid sequencing. The sample was applied to SDS–PAGE and electrophoretically transferred at 7·5 V/cm for 10 hr to Problot membranes (PE Applied Biosystems, Foster City, CA) in transfer buffer (10 mm 3-cyclohexylamino-1-propanesulfonic acid and 10% (v/v) methanol (pH 11)). Protein bands were visualized with Coomassie Brilliant Blue G250 and bands appearing at ∼53 000 MW were cut out and sequenced on an Applied Biosystems 494 A Procise protein sequencer.

Saccharide binding specificity

A solid-phase mannan binding assay was performed by coating microtiter wells (Polysorp; Nunc, Roskilde, Denmark) overnight at 4° with 100 ng mannan (Sigma-Aldrich) in 100 µl buffer made of 15 mm Na2CO3, 35 mm NaHCO3, 30 mm NaN3, pH 7·4. Plates were emptied and incubated for 2 hr at room temperature in TBS/Tw/Ca2+ (10 mm Tris, 150 mm NaCl, 1 mm CaCl2, 0·01% Tween 20, pH 7·4) with 0·1% (w/v) human serum albumin (HSA) (Sigma-Aldrich) and washed in TBS/Tw/Ca2+. Twofold dilutions of 10 µg/ml of pSP-D in TBS/Tw/EDTA (TBS/Tw with 2 mm EDTA) or TBS/Tw/Ca2+ were then added to the wells. The solutions were mixed on a shaking platform and incubated overnight at 4°. The wells were washed in TBS/Tw/Ca2+ and incubated at room temperature for 2 hr with rabbit anti-pSP-D antiserum diluted 1/2000 in TBS/Tw/Ca2+. Wells were washed in TBS/Tw/Ca2+ and incubated with alkaline-phosphatase conjugated goat anti-rabbit-immunoglobulin IgG (Sigma-Aldrich) diluted 1/1000 in TBS/Tw/Ca2+. After incubation at room temperature for 3 hr, wells were washed with TBS/Tw/Ca2+ and developed with para-nitrophenyl phosphate. The absorbance was read at 405 nm by means of a enzyme-linked immunosorbent assay (ELISA) reader (SLT-Labinstruments, Vienna, Austria).

A solid-phase saccharide competition assay was performed for analysing saccharide binding specificity. Microtitre wells were coated with mannan, as described, and dilutions of monosaccharides in 50 µl TBS/Tw/Ca2+ were added in duplicate to the wells. Negative and positive controls consisting of TBS/Tw/EDTA or TBS/Tw/Ca2+, both without monosaccharides, were included. Purified pSP-D at 1·25 µg/ml TBS/Tw/Ca2+ was then added in duplicate 50 µl-volumes. The solutions were mixed on a shaking platform and incubated overnight at 4°. The saccharides tested comprised N-acetyl-d-glucosamine (GlcNAc), N-acetyl-d-mannosamine (ManNAc), maltose (Mal), d-glucose (Glc), d-fucose (Fuc) and d-galactose (Gal) (Sigma-Aldrich). All were tested at concentrations ranging from 0·78 mm to 100 mm. Detection of bound pSP-D was done as described above.

Generation of antibodies against pSP-D

BALB/c female mice were immunized by subcutaneous injections with 25 µg purified pSP-D emulsified with Freund's incomplete adjuvant (Sigma-Aldrich) in 0·25 ml 0·1 m sodium phosphate buffer (phosphate-buffered saline, PBS), pH 7·4. The injections were repeated at intervals of 14 days and mice with a high specific antibody titre were finally boosted intraperitoneally with 20 µg antigen in 0·2 ml PBS. Mice with a high antibody titre against pSP-D, as determined by direct ELISA on SP-D–coated microtitre plates, were selected for fusion of spleen cells. Fusions and screening of hybridoma clones were performed as described previously.34 Briefly, splenocytes were obtained and fused with P3 X63 Ag.8.653 murine myeloma cells (American Type Culture Collection, Rockville, MD). Hybridoma supernatants were tested by an indirect ELISA, using 0·5 µg/ml purified pSP-D in PBS as coating antigen, and positive clones were recloned two or three times. Nine clones were obtained and their specificity were tested by Western blotting. Hybridoma mAb 1.7 was selected for immunohistochemistry analysis and used after purification on protein A agarose (Kem-En-Tec, Copenhagen, Denmark) as previously described35 and after isotyping (Zymed kit, Zymed Laboratories, South San Francisco, CA) according to the protocol recommended by the manufacturer.

Rabbits were immunized subcutaneously with 25 µg of purified pSP-D in Freund's complete adjuvant. The following monthly boosts were done, with the same antigen amount in Freund's incomplete adjuvant as for the production of mAbs, and antisera were collected 14 days after a boost. The polyclonal antibody (pAb) was purified analogously to the mAb on protein A agarose.

Immunohistochemistry

The localization of pSP-D immunoreactivity was investigated using mAb 1.7 on 49 different organ/tissue samples from two approximately 5-month-old and clinically healthy crossbreed (Danish Landrace, Yorkshire, Duroc) slaughter pigs, one of each gender. The organ/tissue samples were lung (cranial and caudal lobes), tracheobronchial lymph node, trachea, larynx, nasal septum (rostral and caudal), Eustachian tube, tympanic bulla, tonsil of the soft palate, buccal mucous membrane, mandibular gland, parotic gland, parotic duct, tongue (corpus), oesophagus, stomach (pars cardiaca, fundus and pylorica), pancreas, common bile duct, pancreatic duct, duodenum, jejunum, ileum, cecum (corpus), colon (gyri centrifugale), rectum, spleen, liver, gall bladder, kidney, adrenal gland, ureter, urinary bladder, urethra, skin (thigh), lacrimal gland (dorsolateral), nasal planum glands, carpeal glands, cerminous glands, thyroid gland, thymus, heart (left ventricular wall), pericardium, synovium (knee joint), subiliacal lymph node, brain (frontal hemisphere), mammary gland (juvenile tissue), vagina, uterus (cervix, corpus and cornu), testicle and ovary.

Tissue samples were fixed for 24–36 hr in 10% (v/v) neutral-buffered formalin, dehydrated, paraffin wax-embedded, sectioned (2–5 µm) and mounted on Superfrost®Plus slides (Merck Eurolab, Albertslund, Denmark). After 15 min dewaxing at 60° the sections were rehydrated in xylene, graded alcohols (99–70% v/v ethanol) and tap water. Endogenous peroxidase activity was blocked with 0·6% H2O2 for 15 min. Epitopes were retrieved by microwave oven heating (700 W) for 2 × 5 min in a buffer containing 0·01 m Tris-base, 0·0005 m EGTA (ethylene glycol-bis(α-aminoethyl ether)-N,N,N,N-tetraacetic acid; Sigma-Aldrich), pH 8 and leaving the sections in the hot buffer for 15 min.

The sections were incubated overnight at 4° with 8·4 µg/ml mAb 1.7 in TBS, 1% bovine serum albumin and a two-layer polymer enhancing immunoperoxidase technique PowerVision + (PV +, Immunovision Technologies, Springdale, AR) was used for signal amplification and detection according to the manufacturer's instructions. Immunostaining was followed by a brief nuclear counterstaining in Mayers haematoxylin and mounted with glycerol-gelatine. Unless otherwise stated, the sections were washed 3 × 5 min in TBS, pH 7·6 between each step and all steps were carried out at room temperature in a moist chamber.

Sections of porcine lung, with strong SP-D immunoreactivity, were included in each test round as a positive staining control, and only rounds with immunoreactivity in control slides were regarded valid. Negative staining controls of all organs/tissues were performed by substituting mAb 1.7 with TBS or an irrelevant monoclonal mouse IgG1 antibody (DAKOCytomation) of identical concentration.

Results

Maltose affinity chromatography and IgM affinity chromatography

Porcine SP-D was purified by maltose affinity chromatography using a MnCl2 step gradient for elution. Contaminating IgM was removed by IgM affinity chromatography. Purified pSP-D showed an approximate molecular mass of ∼53 000 MW in the reduced state, with a weak band seen at ∼51 000 MW, and a molecular mass of ∼138 000 MW unreduced state (Fig. 1)

Figure 1.

SDS–PAGE analysis of maltose and IgM affinity chromatography purified pSP-D. pSP-D migrates under reducing conditions (lane a) as a ∼53 000 MW double band and unreduced pSP-D (lane b) migrates as a ∼138 000 MW band. A 4–20% (w/v) gradient gel and MagicMark molecular weight standard was used.

Enzyme digestion with collagenase and N-glycosidase

When purified pSP-D (Fig. 2, lane a) was treated with collagenase in the presence of calcium (Fig. 2, lane d) a collagen-resistant fragment appeared with a molecular mass of ∼24 000 MW. The digestion could be inhibited by addition of EDTA (Fig. 2, lane c). Porcine SP-D was also modified by N-deglycosylation and the N-deglycosylated form of the protein appeared with a molecular mass of ∼48 000 MW in the reduced state (Fig. 2, lane b). Furthermore the collagenase-resistant fragment was sensitive to N-deglycosylation reducing the molecular mass from ∼24 000 MW to ∼21 000 MW (Fig. 2, lane e). The additional bands seen in lanes b, d and e are the result of the presence of collagenase and/or PNGase.

Figure 2.

Collagenase digested and N-deglycosylated pSP-D analysed by reducing SDS–PAGE. As a positive control pSP-D was analysed (lane a). The N-deglycosylated form of the protein appeared as a ∼48 000 MW band on SDS–PAGE (lane b). pSP-D was collagenase sensitive in the presence of Ca2+ showing a fragment of ∼24 000 MW (lane d) and collagenase insensitive in the presence of EDTA (lane c). Furthermore the collagenase-resistant fragment was sensitive to N-deglycosylation leaving a ∼21 000 MW band (lane e). The additional bands in lanes b, d and e are caused by the presence of collagenase and/or PNGase. A 4–20% (w/v) gradient gel and MagicMark molecular weight standard was used.

N-terminal amino acid sequence

The sequence of the first amino acids of the upper and lower band, in the reduced pSP-D preparation, was determined (Fig. 3). The sequences showed 100% identity with the amino acid sequence predicted from cDNA clones of porcine SP-D.25 The sequence of the lower band showed to be identical, to the sequence found in the upper band from residue 7 onwards. This observation is consistent with the lower band being derived from the upper band by limited proteolysis taking place at the Tyr6Ser7 bond, with the consequent loss of a six-residue long fragment. The sequence showed high similarity to the N-terminal sequences of SP-D from other species (Fig. 3).31,36–39 The conserved cysteine residues at position 15 and 20 were not determined.

Figure 3.

The sequence of the first amino acids of the upper (pSP-D1) and lower (pSP-D2) band, in the reduced pSP-D preparation, was determined. The N-terminal sequence of pSP-D is compared with a sequence predicted from cDNA (pSP-DcDNA),25 and aligned with equine (eSP-D),36 bovine (bSP-D),31 mouse (mSP-D),37 human (hSP-D)38 and rat (rSP-D)39 N-terminal sequences of SP-D. –: denote unidentified residues.

Saccharide binding specificity

The binding of purified pSP-D to solid-phase mannan was dose-dependent, saturable and dependent on the presence of Ca2+(Fig. 4). The relative potencies of a number of mono- and disaccharides in inhibiting the binding of pSP-D to mannan were estimated. The disaccharide maltose was the most potent inhibitor followed by glucose > galactose > ManNAc > fucose > GlcNAc (Table 1). GlcNAc was shown to be approximately 10 times less potent than maltose in inhibition of the binding of pSP-D to mannan (Table 1).

Figure 4.

Solid-phase mannan binding assay. The binding of purified pSP-D to solid-phase mannan was dose-dependent, saturable and dependent on the presence of Ca2+, as the depletion of Ca2+ with EDTA very efficiently inhibited the binding.

Table 1.  Saccharide selectivity of porcine SP-D
SaccharidespSP-D I50
  1. pSP-D I50 is the concentration (mm) of mono- or disaccharides required to give 50% inhibition of binding of pSP-D to mannan.

  2. The values in parentheses denote the I50 concentrations relative to maltose.

Maltose3·1 (1·0)
Glucose5·0 (1·6)
Galactose10·2 (3·3)
ManNAc12·2 (3·9)
Fucose23·3 (7·5)
GlcNAc33·6 (10·8)

Monoclonal antibody against pSP-D

A panel of nine monoclonal anti-pSP-D antibodies were generated and mAb 1.7 (IgG1, kappa) was selected for immunohistochemistry on normal porcine lung tissue.

Figure 5 shows the specificity of mAb 1.7 tested on Western blotting of a reduced and unreduced pSP-D preparation. The same reactivity was seen when purified SP-D was mixed with BAL and when using BAL alone (results not shown). The antibody was found to react with the 53 000 MW band and the 51 000 MW band in the reduced state. In the non-reduced state mAb 1.7 reacted with bands corresponding to a monomer (apparent molecular weight of ∼50 000 MW), a dimer (apparent molecular weight of ∼100 000 MW) and higher oligomers with apparent molecular mass of ∼300 000 MW. In addition a band of ∼31 000 MW was seen, presumably representing a fragment of SP-D.

Figure 5.

The specificity of the antibody used for immunohistochemical analysis (mAb 1.7) analysed by Western blotting. Purified pSP-D was run either in the reduced (lane 1) or unreduced (lane 2) states. Mark 12TM molecular weight standard was used.

Immunohistochemical analysis of pSP-D

SP-D immunoreacticity was present in the porcine lung, small intestine and dorsolateral lacrimal gland. In the lung, strong and specific reactivity was seen in the serous cells and in the lumen of mixed bronchial submucosal glands, while the bronchial epithelium showed no immunoreactivity (Fig. 6a). Strong staining was also seen in epithelial cells of the bronchioles, present as an apical and often granular staining in non-ciliated bronchiolar epithelial cells, also known as Clara cells (Fig. 6b). The immunoreactive cells increased in number from the proximal to the distal part of the bronchiolar tree, leaving many cells unstained in the proximal parts (Fig. 6c). In the distal bronchioles, specially at the bronchiolar–alveolar junctions, almost all cells were pSP-D immunoreactive (Fig. 6d). Generally, the alveolar type II cells showed moderate (Figs 6c, e) to weak diffuse intracellular staining intensity. Extracellular luminal staining along the epithelial surface of the alveolar septae was generally not seen.

Figure 6.

Porcine surfactant protein D in the lung of an adult pig. (a) Strong pSP-D immunoreactivity in serous cells of the bronchial submucosal glands. Note absent immunoreactivity of the bronchial epithelium and the mucous cells of the bronchial submucosal glands. Bar = 24 (insert) or 48 µm (b) Strong apical pSP-D immunoreactivity in bronchiolar Clara cells with their characteristic dome-shaped apex. The Clara cells are adjacent to ciliated epithelia cells. Bar = 10 µm (c) Distal bronchiole with strong immunoreactivity in the no-ciliated low cuboidal Clara cells and a moderate, diffuse intracellular staining intensity in the adjacent alveolar type II cells. Bar = 24 µm (d) Porcine SP-D immunoreactive Clara cells at the bronchiolar–alveolar junctions, lining almost the entire bronchiole. Bar = 24 µm (e) Diffuse intracellular staining in the alveolar type II cells. Bar = 24 µm. Sections detected with two-layer polymer, immunoperoxidase technique and counterstained with Mayers's haematoxylin as described in Materials and Methods.

Weak, but significant and frequent, staining was seen in cells of the intestinal glands (crypt of Lieberkühn) in the duodenum, jejunum and ileum (Figs 7a, b). Epithelial cells of the intestinal villi (Fig. 7a) and mucin in the goblet cells, of both the intestinal glands and villi, showed no immunoreactivity (Fig. 7b). No immunoreactivity was found in the rest of the gastrointestinal tract.

Figure 7.

Porcine SP-D in the small intestine and in the lacrimal gland. (a) Weak, diffuse intracellular immunoreactivity in the epithelial cells of the intestinal glands (crypt of Lieberkühn) in the ileum. Bar = 48 µm (b) No immunoreactivity was seen in epithelial cells of the intestinal villus or mucin in the goblet cells Bar = 24 µm. (c) and (d) Moderate to strong immunoreactivity was seen intracellularly in serous cells of the lacrimal gland. Bar = 10 µm. Sections detected with two-layer polymer, immunoperoxidase technique and counterstained with Mayers's haematoxylin as described in Materials and Methods.

In the dorsolateral lacrimal gland moderate to strong, but infrequent immunoreactivity was seen in serous cells of the mixed gland (Figs 7c, d).

No staining was seen substituting anti-pSP-D mAb 1.7 with TBS or an irrelevant antibody of identical isotype and concentration (not shown).

Discussion

Here we describe the purification of pSP-D by affinity chromatography on maltose-agarose combined with depletion affinity chromatography on anti-IgM-Sepharose. The pSP-D polypeptide chain has an apparent molecular mass of ∼53 000 MW on SDS–PAGE comparable with a 50 000 MW porcine SP-D, recently described by van Eijk et al.26 A second polypeptide chain of ∼51 000 MW is seen in the pSP-D preparations. This chain can partially be explained by a limited proteolysis taking place at a Tyr-Ser bond near the end of the intact ∼53 000 MW chain, a similar proteolysis previously described in conglutinin40 and CL-43.41 The SDS–PAGE pattern under non-reducing conditions revealed a main band of ∼138 000 MW. This band probably consists of three ∼53 000 MW chains held together by disulphide bonds. Minor bands with molecular mass over 200 000 MW were also seen and these bands most likely represent higher oligomeric forms of the pSP-D. Collagenase treatment of pSP-D produced a single collagenase resistant fragment from the ∼53 000 MW and ∼51 000 MW chains. The molecular mass of ∼24 000 of this collagenase resistant fragment is similar to that observed for the collagenase-resistant, C-terminal, domains of other collectins.26,31,36,38 Both the ∼53 000 MW monomer and the ∼24 000 MW collagenase-resistant C-terminal fragment were affected by N-deglycosylation leaving a ∼48 000 MW and a ∼21 000 MW band on SDS–PAGE, respectively. This indicates the presence of an N-glycosylation site, glycosylated with ∼3000–5000 MW saccharide, probably located in the CRD region in agreement with what was previously shown by van Eijk et al.26 Both human and bovine SP-D was found to be O- as well as N-glycosylated in the collagen region31 and Mason et al.42 found a human 50 000 MW variant to be extensively O-glycosylated in the N-terminal region. Porcine SP-D thereby differs from SP-D from other species in being larger and being the only SP-D known to be glycosylated in the CRD region. We analysed the saccharide binding specificity of pSP-D in an inhibition assay and found it similar to the specificity of rat43 and human38 SP-D. The best inhibitor was maltose followed by glucose, galactose, fucose and GlcNAc. The disaccharide maltose was shown to be approximately 10 times more potent as inhibitor of binding compared to GlcNAc. The presence of the N-glycosylation in the CRD region in pSP-D does not seem to alter the saccharide specificity significantly compared with SP-D in other species. The CRD located N-carbohydrate moiety may, however, influence the interaction of pSP-D with micro-organisms as removal of this structure reduced the Ca2+-dependent inhibitory effect of pSP-D on Influenza A virus haemagglutination.26 The relative size of the purified protein, its sensitivity to collagenase and N-deglycosylation, its dose and calcium dependent binding to mannan, its carbohydrate specificity and the N-terminal amino acid sequence confirmed that the isolated protein was indeed pSP-D.

This biochemically characterized pool of purified protein was used to generate a total of nine monoclonal antibodies directed against pSP-D. One monoclonal antibody (mAb 1.7), which showed specific staining for purified pSP-D, and purified pSP-D + BAL and BAL alone (results not shown) in the reduced as well as in the unreduced state, was chosen for immunohistochemical localization of pSP-D. By using this antibody for immunolocalization, pSP-D was specifically demonstrated in the porcine lung, small intestine and dorsolateral lacrimal gland. The identification of pSP-D immunoreactive cells of the lung as serous glandular cells, Clara cells and alveolar type II cells was based on cellular morphology and distribution. The pSP-D immunoreactive Clara cells were identified by their lack of cilia and their dome-shaped apex, which is the site for the discharge of granular contents.44 Furthermore, the largest number of pSP-D immunoreactive epithelial cells being observed in the distal bronchioles and a proximally decreasing number of these cells, coincides with the decreasing number of Clara cells in the more proximal airways.45 The apical localization of pSP-D in Clara cells is similar to the specific labelling of rat SP-D in apical secretory granules in Clara cells, previously described.46,47 The localization of SP-D to alveolar type II cells and Clara cells has been well documented in human,48 rat46,47 and mouse.49 Alveolar type II cells have been referred to as the major48 or only50 site of pulmonary SP-D localization. In pigs, Clara cells stained more intensely than alveolar type II cells. Other techniques with lower sensitivity, than the described immunoperoxidase technique, were in general unable to detect pSP-D in alveolar type II cells, while Clara cells remained strongly stained (not shown). In type II cells SP-D, and other surfactant proteins, are stored in lamellar bodies, while in Clara cells the protein is stored in secretory granules.46 The difference in immunoreactivity could be caused by a lower content of pSP-D in the type II cells than in Clara cells, or a difference in detectability resulting from storage in different compartments.

In the distal bronchioles, specially at the bronchiolar-alveolar junctions, almost all cells were pSP-D immunoreactive. Interestingly, the initial injury in bronchopneumonia is centred in the distal respiratory tract, at the bronchiolar–alveolar junctions. These are the major sites of deposition of small particles capable of reaching deep lung.51 The distal bronchioles are not protected by the mucous blanket of larger airways or by an effective alveolar macrophage system51 and it therefore seems ideal with a high level of SP-D at this location, lining the mucosa for prevention of microbial colonization and invasion.

Porcine SP-D was also found in the serous cells and in the luminal part of bronchial submucosal glands, and as SP-D immunoreactivity is absent from the bronchial epithelium, secretion from these glands might be a source of pSP-D in the bronchi.

Extrapulmonary localization of SP-D has been detected in different species in localizations as different as the salivary gland, trachea, heart, pancreas, stomach, small intestine, colon, mesentery, brain, uterus, ovary, kidney, and many more.25,37,46–50,52–54 Recent Northern blot analysis of the tissue distribution of porcine SP-D mRNA, using a porcine SP-D CRD probe, showed the presence of strong signal in lung tissue and weaker signal in duodenum, jejunum, ileum, and in addition ileal mucosa, but no signal was found in the mucosa extracted from the duodenum and jejunum.25 We demonstrate here the presence of pSP-D in the intestinal glands (crypts of Lieberkühn) located in the mucosa, in all parts of the small intestine. Immunolocalization of SP-D specific to the intestinal glands (crypts of Lieberkühn) in the small intestine has also been described in human.50 Porcine SP-D immunoreactivity has previously been found in the Eustachian tube using a polyclonal antibody.53 We were not able to confirm this localization for pSP-D, which might be caused by differences in the applied antibodies, sensitivity of the methods applied or individual difference in the tissue expression of the protein.

The localization of pSP-D in serous cells in the porcine lacrimal gland, a gland in which SP-D detection previously has been seen in human48,50 and mouse49 was however, demonstrated. The low number of immunoreactive serous cells found in the lacrimal gland, coincides with a low number of serous cells caused by a predominance of mucous cells in the porcine lacrimal gland.55

The common features of Clara cells, alveolar type II cells, intestinal, lacrimal and bronchial submucosal gland cells are their important secretory functions.56 The localization of pSP-D in these secretory cells in the lung and in serous secretory cells in the bronchial, intestinal and lacrimal glands, indicates a constitutive local production of pSP-D. A constant presence of pSP-D strongly supports an active function in the innate mucosal defence system, against invading pathogenic micro-organisms.

Acknowledgements

This work was kindly supported by the Novo Nordisk Foundation. We thank Ida Tornoe, Vivi A. Moeller, Heidi Pedersen, Regina Lund, Lisbet Kioerboe, Betina Andersen, Hanne H. Moeller and Dennis Brok for excellent technical assistance.

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