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Keywords:

  • immunology;
  • innate immunity;
  • collectins;
  • porcine;
  • surfactant protein D;
  • immunolocalization

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Sampling and preparation of bronchoalveolar lavage (BAL)
  6. Maltose affinity chromatography
  7. Immunoglobulin M (IgM) affinity chromatography
  8. SDS–PAGE and Western blotting
  9. Collagenase digestion
  10. N-deglycosylation
  11. N-terminal amino acid sequencing
  12. Saccharide binding specificity
  13. Generation of antibodies against pSP-D
  14. Immunohistochemistry
  15. Results
  16. Maltose affinity chromatography and IgM affinity chromatography
  17. Enzyme digestion with collagenase and N-glycosidase
  18. N-terminal amino acid sequence
  19. Saccharide binding specificity
  20. Monoclonal antibody against pSP-D
  21. Immunohistochemical analysis of pSP-D
  22. Discussion
  23. Acknowledgements
  24. References

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

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Sampling and preparation of bronchoalveolar lavage (BAL)
  6. Maltose affinity chromatography
  7. Immunoglobulin M (IgM) affinity chromatography
  8. SDS–PAGE and Western blotting
  9. Collagenase digestion
  10. N-deglycosylation
  11. N-terminal amino acid sequencing
  12. Saccharide binding specificity
  13. Generation of antibodies against pSP-D
  14. Immunohistochemistry
  15. Results
  16. Maltose affinity chromatography and IgM affinity chromatography
  17. Enzyme digestion with collagenase and N-glycosidase
  18. N-terminal amino acid sequence
  19. Saccharide binding specificity
  20. Monoclonal antibody against pSP-D
  21. Immunohistochemical analysis of pSP-D
  22. Discussion
  23. Acknowledgements
  24. References

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.

Sampling and preparation of bronchoalveolar lavage (BAL)

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Sampling and preparation of bronchoalveolar lavage (BAL)
  6. Maltose affinity chromatography
  7. Immunoglobulin M (IgM) affinity chromatography
  8. SDS–PAGE and Western blotting
  9. Collagenase digestion
  10. N-deglycosylation
  11. N-terminal amino acid sequencing
  12. Saccharide binding specificity
  13. Generation of antibodies against pSP-D
  14. Immunohistochemistry
  15. Results
  16. Maltose affinity chromatography and IgM affinity chromatography
  17. Enzyme digestion with collagenase and N-glycosidase
  18. N-terminal amino acid sequence
  19. Saccharide binding specificity
  20. Monoclonal antibody against pSP-D
  21. Immunohistochemical analysis of pSP-D
  22. Discussion
  23. Acknowledgements
  24. References

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

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Sampling and preparation of bronchoalveolar lavage (BAL)
  6. Maltose affinity chromatography
  7. Immunoglobulin M (IgM) affinity chromatography
  8. SDS–PAGE and Western blotting
  9. Collagenase digestion
  10. N-deglycosylation
  11. N-terminal amino acid sequencing
  12. Saccharide binding specificity
  13. Generation of antibodies against pSP-D
  14. Immunohistochemistry
  15. Results
  16. Maltose affinity chromatography and IgM affinity chromatography
  17. Enzyme digestion with collagenase and N-glycosidase
  18. N-terminal amino acid sequence
  19. Saccharide binding specificity
  20. Monoclonal antibody against pSP-D
  21. Immunohistochemical analysis of pSP-D
  22. Discussion
  23. Acknowledgements
  24. References

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

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Sampling and preparation of bronchoalveolar lavage (BAL)
  6. Maltose affinity chromatography
  7. Immunoglobulin M (IgM) affinity chromatography
  8. SDS–PAGE and Western blotting
  9. Collagenase digestion
  10. N-deglycosylation
  11. N-terminal amino acid sequencing
  12. Saccharide binding specificity
  13. Generation of antibodies against pSP-D
  14. Immunohistochemistry
  15. Results
  16. Maltose affinity chromatography and IgM affinity chromatography
  17. Enzyme digestion with collagenase and N-glycosidase
  18. N-terminal amino acid sequence
  19. Saccharide binding specificity
  20. Monoclonal antibody against pSP-D
  21. Immunohistochemical analysis of pSP-D
  22. Discussion
  23. Acknowledgements
  24. References

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

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Sampling and preparation of bronchoalveolar lavage (BAL)
  6. Maltose affinity chromatography
  7. Immunoglobulin M (IgM) affinity chromatography
  8. SDS–PAGE and Western blotting
  9. Collagenase digestion
  10. N-deglycosylation
  11. N-terminal amino acid sequencing
  12. Saccharide binding specificity
  13. Generation of antibodies against pSP-D
  14. Immunohistochemistry
  15. Results
  16. Maltose affinity chromatography and IgM affinity chromatography
  17. Enzyme digestion with collagenase and N-glycosidase
  18. N-terminal amino acid sequence
  19. Saccharide binding specificity
  20. Monoclonal antibody against pSP-D
  21. Immunohistochemical analysis of pSP-D
  22. Discussion
  23. Acknowledgements
  24. References

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.

N-terminal amino acid sequencing

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Sampling and preparation of bronchoalveolar lavage (BAL)
  6. Maltose affinity chromatography
  7. Immunoglobulin M (IgM) affinity chromatography
  8. SDS–PAGE and Western blotting
  9. Collagenase digestion
  10. N-deglycosylation
  11. N-terminal amino acid sequencing
  12. Saccharide binding specificity
  13. Generation of antibodies against pSP-D
  14. Immunohistochemistry
  15. Results
  16. Maltose affinity chromatography and IgM affinity chromatography
  17. Enzyme digestion with collagenase and N-glycosidase
  18. N-terminal amino acid sequence
  19. Saccharide binding specificity
  20. Monoclonal antibody against pSP-D
  21. Immunohistochemical analysis of pSP-D
  22. Discussion
  23. Acknowledgements
  24. References

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

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Sampling and preparation of bronchoalveolar lavage (BAL)
  6. Maltose affinity chromatography
  7. Immunoglobulin M (IgM) affinity chromatography
  8. SDS–PAGE and Western blotting
  9. Collagenase digestion
  10. N-deglycosylation
  11. N-terminal amino acid sequencing
  12. Saccharide binding specificity
  13. Generation of antibodies against pSP-D
  14. Immunohistochemistry
  15. Results
  16. Maltose affinity chromatography and IgM affinity chromatography
  17. Enzyme digestion with collagenase and N-glycosidase
  18. N-terminal amino acid sequence
  19. Saccharide binding specificity
  20. Monoclonal antibody against pSP-D
  21. Immunohistochemical analysis of pSP-D
  22. Discussion
  23. Acknowledgements
  24. References

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

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Sampling and preparation of bronchoalveolar lavage (BAL)
  6. Maltose affinity chromatography
  7. Immunoglobulin M (IgM) affinity chromatography
  8. SDS–PAGE and Western blotting
  9. Collagenase digestion
  10. N-deglycosylation
  11. N-terminal amino acid sequencing
  12. Saccharide binding specificity
  13. Generation of antibodies against pSP-D
  14. Immunohistochemistry
  15. Results
  16. Maltose affinity chromatography and IgM affinity chromatography
  17. Enzyme digestion with collagenase and N-glycosidase
  18. N-terminal amino acid sequence
  19. Saccharide binding specificity
  20. Monoclonal antibody against pSP-D
  21. Immunohistochemical analysis of pSP-D
  22. Discussion
  23. Acknowledgements
  24. References

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

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Sampling and preparation of bronchoalveolar lavage (BAL)
  6. Maltose affinity chromatography
  7. Immunoglobulin M (IgM) affinity chromatography
  8. SDS–PAGE and Western blotting
  9. Collagenase digestion
  10. N-deglycosylation
  11. N-terminal amino acid sequencing
  12. Saccharide binding specificity
  13. Generation of antibodies against pSP-D
  14. Immunohistochemistry
  15. Results
  16. Maltose affinity chromatography and IgM affinity chromatography
  17. Enzyme digestion with collagenase and N-glycosidase
  18. N-terminal amino acid sequence
  19. Saccharide binding specificity
  20. Monoclonal antibody against pSP-D
  21. Immunohistochemical analysis of pSP-D
  22. Discussion
  23. Acknowledgements
  24. References

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.

Maltose affinity chromatography and IgM affinity chromatography

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Sampling and preparation of bronchoalveolar lavage (BAL)
  6. Maltose affinity chromatography
  7. Immunoglobulin M (IgM) affinity chromatography
  8. SDS–PAGE and Western blotting
  9. Collagenase digestion
  10. N-deglycosylation
  11. N-terminal amino acid sequencing
  12. Saccharide binding specificity
  13. Generation of antibodies against pSP-D
  14. Immunohistochemistry
  15. Results
  16. Maltose affinity chromatography and IgM affinity chromatography
  17. Enzyme digestion with collagenase and N-glycosidase
  18. N-terminal amino acid sequence
  19. Saccharide binding specificity
  20. Monoclonal antibody against pSP-D
  21. Immunohistochemical analysis of pSP-D
  22. Discussion
  23. Acknowledgements
  24. References

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)

image

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.

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Enzyme digestion with collagenase and N-glycosidase

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Sampling and preparation of bronchoalveolar lavage (BAL)
  6. Maltose affinity chromatography
  7. Immunoglobulin M (IgM) affinity chromatography
  8. SDS–PAGE and Western blotting
  9. Collagenase digestion
  10. N-deglycosylation
  11. N-terminal amino acid sequencing
  12. Saccharide binding specificity
  13. Generation of antibodies against pSP-D
  14. Immunohistochemistry
  15. Results
  16. Maltose affinity chromatography and IgM affinity chromatography
  17. Enzyme digestion with collagenase and N-glycosidase
  18. N-terminal amino acid sequence
  19. Saccharide binding specificity
  20. Monoclonal antibody against pSP-D
  21. Immunohistochemical analysis of pSP-D
  22. Discussion
  23. Acknowledgements
  24. References

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.

image

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.

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N-terminal amino acid sequence

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Sampling and preparation of bronchoalveolar lavage (BAL)
  6. Maltose affinity chromatography
  7. Immunoglobulin M (IgM) affinity chromatography
  8. SDS–PAGE and Western blotting
  9. Collagenase digestion
  10. N-deglycosylation
  11. N-terminal amino acid sequencing
  12. Saccharide binding specificity
  13. Generation of antibodies against pSP-D
  14. Immunohistochemistry
  15. Results
  16. Maltose affinity chromatography and IgM affinity chromatography
  17. Enzyme digestion with collagenase and N-glycosidase
  18. N-terminal amino acid sequence
  19. Saccharide binding specificity
  20. Monoclonal antibody against pSP-D
  21. Immunohistochemical analysis of pSP-D
  22. Discussion
  23. Acknowledgements
  24. References

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.

image

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.

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Saccharide binding specificity

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Sampling and preparation of bronchoalveolar lavage (BAL)
  6. Maltose affinity chromatography
  7. Immunoglobulin M (IgM) affinity chromatography
  8. SDS–PAGE and Western blotting
  9. Collagenase digestion
  10. N-deglycosylation
  11. N-terminal amino acid sequencing
  12. Saccharide binding specificity
  13. Generation of antibodies against pSP-D
  14. Immunohistochemistry
  15. Results
  16. Maltose affinity chromatography and IgM affinity chromatography
  17. Enzyme digestion with collagenase and N-glycosidase
  18. N-terminal amino acid sequence
  19. Saccharide binding specificity
  20. Monoclonal antibody against pSP-D
  21. Immunohistochemical analysis of pSP-D
  22. Discussion
  23. Acknowledgements
  24. References

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).

image

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.

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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

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Sampling and preparation of bronchoalveolar lavage (BAL)
  6. Maltose affinity chromatography
  7. Immunoglobulin M (IgM) affinity chromatography
  8. SDS–PAGE and Western blotting
  9. Collagenase digestion
  10. N-deglycosylation
  11. N-terminal amino acid sequencing
  12. Saccharide binding specificity
  13. Generation of antibodies against pSP-D
  14. Immunohistochemistry
  15. Results
  16. Maltose affinity chromatography and IgM affinity chromatography
  17. Enzyme digestion with collagenase and N-glycosidase
  18. N-terminal amino acid sequence
  19. Saccharide binding specificity
  20. Monoclonal antibody against pSP-D
  21. Immunohistochemical analysis of pSP-D
  22. Discussion
  23. Acknowledgements
  24. References

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.

image

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.

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Immunohistochemical analysis of pSP-D

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Sampling and preparation of bronchoalveolar lavage (BAL)
  6. Maltose affinity chromatography
  7. Immunoglobulin M (IgM) affinity chromatography
  8. SDS–PAGE and Western blotting
  9. Collagenase digestion
  10. N-deglycosylation
  11. N-terminal amino acid sequencing
  12. Saccharide binding specificity
  13. Generation of antibodies against pSP-D
  14. Immunohistochemistry
  15. Results
  16. Maltose affinity chromatography and IgM affinity chromatography
  17. Enzyme digestion with collagenase and N-glycosidase
  18. N-terminal amino acid sequence
  19. Saccharide binding specificity
  20. Monoclonal antibody against pSP-D
  21. Immunohistochemical analysis of pSP-D
  22. Discussion
  23. Acknowledgements
  24. References

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.

image

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.

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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.

image

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.

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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

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Sampling and preparation of bronchoalveolar lavage (BAL)
  6. Maltose affinity chromatography
  7. Immunoglobulin M (IgM) affinity chromatography
  8. SDS–PAGE and Western blotting
  9. Collagenase digestion
  10. N-deglycosylation
  11. N-terminal amino acid sequencing
  12. Saccharide binding specificity
  13. Generation of antibodies against pSP-D
  14. Immunohistochemistry
  15. Results
  16. Maltose affinity chromatography and IgM affinity chromatography
  17. Enzyme digestion with collagenase and N-glycosidase
  18. N-terminal amino acid sequence
  19. Saccharide binding specificity
  20. Monoclonal antibody against pSP-D
  21. Immunohistochemical analysis of pSP-D
  22. Discussion
  23. Acknowledgements
  24. References

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.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Sampling and preparation of bronchoalveolar lavage (BAL)
  6. Maltose affinity chromatography
  7. Immunoglobulin M (IgM) affinity chromatography
  8. SDS–PAGE and Western blotting
  9. Collagenase digestion
  10. N-deglycosylation
  11. N-terminal amino acid sequencing
  12. Saccharide binding specificity
  13. Generation of antibodies against pSP-D
  14. Immunohistochemistry
  15. Results
  16. Maltose affinity chromatography and IgM affinity chromatography
  17. Enzyme digestion with collagenase and N-glycosidase
  18. N-terminal amino acid sequence
  19. Saccharide binding specificity
  20. Monoclonal antibody against pSP-D
  21. Immunohistochemical analysis of pSP-D
  22. Discussion
  23. Acknowledgements
  24. References
  • 1
    Curstedt T, Jornvall H, Robertson B, Bergman T, Berggren P. Two hydrophobic low-molecular-mass protein fractions of pulmonary surfactant. Characterization and biophysical activity. Eur J Biochem 1987; 168: 25562.
  • 2
    Holmskov U, Malhotra R, Sim RB, Jensenius JC. Collectins: collagenous C-type lectins of the innate immune defense system. Immunol Today 1994; 15: 6774.DOI: 10.1016/0167-5699(94)90136-8
  • 3
    Wright JR, Borchelt JD, Hawgood S. Lung surfactant apoprotein SP-A (26–36 kDa) binds with high affinity to isolated alveolar type II cells. Proc Natl Acad Sci U S A 1989; 86: 54104.
  • 4
    Crouch E, Rust K, Persson A, Mariencheck W, Moxley M, Longmore W. Primary translation products of pulmonary surfactant protein D. Am J Physiol 1991; 260: L247L253.
  • 5
    Kawasaki N, Kawasaki T, Yamashina I. Isolation and characterization of a mannan-binding protein from human serum. J Biochem (Tokyo) 1983; 94: 93747.
  • 6
    Jensenius JC, Laursen SB, Zheng Y, Holmskov U. Conglutinin and CL-43, two collagenous C-type lectins (collectins) in bovine serum. Biochem Soc Trans 1994; 22: 95100.
  • 7
    Holmskov U, Teisner B, Willis AC, Reid KB, Jensenius JC. Purification and characterization of a bovine serum lectin (CL-43) with structural homology to conglutinin and SP-D and carbohydrate specificity similar to mannan-binding protein. J Biol Chem 1993; 268: 101205.
  • 8
    Hansen S, Holm D, Moeller V, Vitved L, Bendixen C, Reid KB, Skjoedt K, Holmskov U. CL-46, a novel collectin highly expressed in bovine thymus and liver. J Immunol 2002; 169: 572634.
  • 9
    Ohtani K, Suzuki Y, Eda S et al. Molecular cloning of a novel human collectin from liver (CL-L1). J Biol Chem 1999; 274: 136819.DOI: 10.1074/jbc.274.19.13681
  • 10
    Ohtani K, Suzuki Y, Eda S et al. The membrane-type collectin CL-P1 is a scavenger receptor on vascular endothelial cells. J Biol Chem 2001; 276: 442228.DOI: 10.1074/jbc.M103942200
  • 11
    Crouch E, Wright JR. Surfactant proteins a and d and pulmonary host defense. Annu Rev Physiol 2001; 63: 52154.
  • 12
    Crouch EC. Surfactant protein-D and pulmonary host defense. Respir Res 2000; 1: 93108.
  • 13
    Holmskov U, Thiel S, Jensenius JC. Collections and ficolins: humoral lectins of the innate immune defense. Annu Rev Immunol 2003; 21: 54778.DOI: 10.1146/annurev.immunol.21.120601.140954
  • 14
    McCormack FX, Whitsett JA. The pulmonary collectins, SP-A and SP-D, orchestrate innate immunity in the lung. J Clin Invest 2002; 109: 70712.DOI: 10.1172/JCI200215293
  • 15
    Wright JR. Host defense functions of pulmonary surfactant. Biol Neonate 2004; 85: 32632.DOI: 10.1159/000078172
  • 16
    Wu H, Kuzmenko A, Wan S, Schaffer L, Weiss A, Fisher JH, Kim KS, McCormack FX. Surfactant proteins A and D inhibit the growth of Gram-negative bacteria by increasing membrane permeability. J Clin Invest 2003; 111: 1589602.DOI: 10.1172/JCI200316889
  • 17
    Ikegami M, Whitsett JA, Jobe A, Ross G, Fisher J, Korfhagen T. Surfactant metabolism in SP-D gene-targeted mice. Am J Physiol Lung Cell Mol Physiol 2000; 279: L468L476.
  • 18
    Brinker KG, Martin E, Borron P, Mostaghel E, Doyle C, Harding CV, Wright JR. Surfactant protein D enhances bacterial antigen presentation by bone marrow-derived dendritic cells. Am J Physiol Lung Cell Mol Physiol 2001; 281: L1453L1463.
  • 19
    Christensen G, Sorensen V, Mousing J. Diseases of swine. In: StrawBE, D'AllaireS, MengelingWL, TaylorDJ, eds. Diseases in Swine, 8th edn. Oxford: Blackwell Science, 1999: 91340.
  • 20
    Van Reeth K, Nauwynck H, Pensaert M. Bronchoalveolar interferon-alpha, tumor necrosis factor-alpha, interleukin-1, and inflammation during acute influenza in pigs: a possible model for humans? J Infect Dis 1998; 177: 10769.
  • 21
    Dinger J, Topfer A, Schaller P, Schwarze R. Functional residual capacity and compliance of the respiratory system after surfactant treatment in premature infants with severe respiratory distress syndrome. Eur J Pediatr 2002; 161: 48590.DOI: 10.1007/s00431-002-0989-6
  • 22
    Spragg RG. Surfactant therapy in acute respiratory distress syndrome. Biol Neonat 1998; 74(Suppl. 1):1520.DOI: 10.1159/000047030
  • 23
    Sun B. Use of surfactant in pulmonary disorders in full-term infants. Curr Opin Pediatr 1996; 8: 1137.
  • 24
    Halliday HL. Synthetic or natural surfactants. Acta Paediatr 1997; 86: 2337.
  • 25
    Van Eijk M, Haagsman HP, Skinner T, Archibald A, Reid KB, Lawson PR, Archibold A. Porcine lung surfactant protein D. complementary DNA cloning, chromosomal localization, and tissue distribution. J Immunol 2000; 164: 144250.
  • 26
    Van Eijk M, Van De Lest CH, Batenburg JJ, Vaandrager AB, Meschi J, Hartshorn KL, Van Golde LM, Haagsman HP. Porcine surfactant protein D is N-glycosylated in its carbohydrate recognition domain and is assembled into differently charged oligomers. Am J Respir Cell Mol Biol 2002; 26: 73947.
  • 27
    Van Eijk M, White MR, Batenburg JJ, Vaandrager AB, Van Golde LM, Haagsman HP, Hartshorn KL. Interactions of Influenza A virus with sialic acids present on porcine surfactant protein D. Am J Respir Cell Mol Biol 2004; 30: 8719.DOI: 10.1165/rcmb.2003-0355OC
  • 28
    Van Eijk M, White MR, Crouch EC, Batenburg JJ, Vaandrager AB, Van Golde LM, Haagsman HP, Hartshorn KL 2003 Porcine pulmonary collectins show distinct interactions with influenza A viruses: role of the N-linked oligosaccharides in the carbohydrate recognition domain. J Immunol 2003; 171: 143140.
  • 29
    Strong P, Kishore U, Morgan C, Lopez BA, Singh M, Reid KB. A novel method of purifying lung surfactant proteins A and D from the lung lavage of alveolar proteinosis patients and from pooled amniotic fluid. J Immunol Methods 1998; 220: 13949.DOI: 10.1016/S0022-1759(98)00160-4
  • 30
    Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 6805.
  • 31
    Leth-Larsen R, Holmskov U, Hojrup P. Structural characterization of human and bovine lung surfactant protein D. Biochem J 1999; 343 Part 3: 64552.DOI: 10.1042/0264-6021:3430645
  • 32
    Heukeshoven J, Dernick R. Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 1985; 6: 10312.
  • 33
    Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets – procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 43504.
  • 34
    Goding JW. Antibody production by hybridomas. J Immunol Methods 1980; 39: 285308.DOI: 10.1016/0022-1759(80)90230-6
  • 35
    Manil L, Motte P, Pernas P, Troalen F, Bohuon C, Bellet D. Evaluation of protocols for purification of mouse monoclonal antibodies. Yield and purity in two-dimensional gel electrophoresis. J Immunol Methods 1986; 90: 2537.DOI: 10.1016/0022-1759(86)90379-0
  • 36
    Hobo S, Ogasawara Y, Kuroki Y, Akino T, Yoshihara T. Purification and biochemical characterization of equine pulmonary surfactant protein D. Am J Vet Res 1999; 60: 36872.
  • 37
    Motwani M, White RA, Guo N, Dowler LL, Tauber AI, Sastry KN. Mouse surfactant protein-D. cDNA cloning, characterization, and gene localization to chromosome 14. J Immunol 1995; 155: 56717.
  • 38
    Lu J, Willis AC, Reid KB. Purification, characterization and cDNA cloning of human lung surfactant protein D. Biochem J 1992; 284(3):795802.
  • 39
    Shimizu H, Fisher JH, Papst P, Benson B, Lau K, Mason RJ, Voelker DR. Primary structure of rat pulmonary surfactant protein D. cDNA and deduced amino acid sequence. J Biol Chem 1992; 267: 18537.
  • 40
    Lu J, Wiedemann H, Holmskov U, Thiel S, Timpl R, Reid KB. Structural similarity between lung surfactant protein D and conglutinin. Two distinct, C-type lectins containing collagen-like sequences. Eur J Biochem 1993; 215: 7939.
  • 41
    Holmskov U, Laursen SB, Malhotra R et al. Comparative study of the structural and functional properties of a bovine plasma C-type lectin, collectin-43, with other collectins. Biochem J 1995; 305(3):88996.
  • 42
    Mason RJ, Nielsen LD, Kuroki Y, Matsuura E, Freed JH, Shannon JM. A 50-kDa variant form of human surfactant protein D. Eur Respir J 1998; 12: 114755.DOI: 10.1183/09031936.98.12051147
  • 43
    Persson A, Chang D, Rust K, Moxley M, Longmore W, Crouch E. Purification and biochemical characterization of CP4 (SP-D), a collagenous surfactant-associated protein. Biochemistry 1989; 28: 63617.
  • 44
    Ross MH, Reith EJ, Romrell LJ. Respiratory system. In: KistK, ed. Histology. A Text and Atlas, 2nd edn. Baltimore: Williams & Wilkins, 2003: 50124.
  • 45
    Plopper CG, Adams DR. Respiratory system. In: DellmannH, BrownEM, eds. Textbook of Veterinary Histology. Philadelphia: Lea & Febiger, 1993: 13652.
  • 46
    Crouch E, Parghi D, Kuan SF, Persson A. Surfactant protein D. subcellular localization in nonciliated bronchiolar epithelial cells. Am J Physiol 1992; 263: L60L66.
  • 47
    Voorhout WF, Veenendaal T, Kuroki Y, Ogasawara Y, Van Golde LM, Geuze HJ. Immunocytochemical localization of surfactant protein D (SP-D) in type II cells, Clara cells, and alveolar macrophages of rat lung. J Histochem Cytochem 1992; 40: 158997.
  • 48
    Madsen J, Kliem A, Tornoe I, Skjodt K, Koch C, Holmskov U. Localization of lung surfactant protein D on mucosal surfaces in human tissues. J Immunol 2000; 164: 586670.
  • 49
    Akiyama J, Hoffman A, Brown C, Allen L, Edmondson J, Poulain F, Hawgood S. Tissue distribution of surfactant proteins A and D in the mouse. J Histochem Cytochem 2002; 50: 9936.
  • 50
    Stahlman MT, Gray ME, Hull WM, Whitsett JA. Immunolocalization of surfactant protein-D (SP-D) in human fetal, newborn, and adult tissues. J Histochem Cytochem 2002; 50: 65160.
  • 51
    Dungworth DL. The respiratory system. In: JubbKVF, KennedyPC, PalmerN, eds. Pathology of Domestic Animals, Vol. 2, 4th edn. San Diego: Academic Press Inc., 1993: 539698.
  • 52
    Fisher JH, Mason R. Expression of pulmonary surfactant protein D in rat gastric mucosa. Am J Respir Cell Mol Biol 1995; 12: 138.
  • 53
    Paananen R, Glumoff V, Hallman M. Surfactant protein A and D expression in the porcine Eustachian tube. FEBS Lett 1999; 452: 1414.DOI: 10.1016/S0014-5793(99)00602-X
  • 54
    Wong CJ, Akiyama J, Allen L, Hawgood S. Localization and developmental expression of surfactant proteins D and A in the respiratory tract of the mouse. Pediatr Res 1996; 39: 9307.
  • 55
    Dellmann H. Eye and ear. In: DellmannH, BrownEM, eds. Textbook of Veterinary Histology. Philadelphia: Lea & Febiger, 2003: 42356.
  • 56
    Dellmann H, Brown EM, eds. Textbook of Veterinary Histology. Philadelphia: Lea & Febiger, 1993.