CHARACTERIZATION OF SURFACTANT PROTEINS IN PORCINE EUSTACHIAN TUBE

REIJAPAANANEN

Department of Paediatrics,University of Oulu

Biocenter Oulu,University of Oulu

OULU 2001

REIJA PAANANEN

CHARACTERIZATION OF SURFACTANT PROTEINS IN PORCINE EUSTACHIAN TUBE

Academic Dissertation to be presented with the assent ofthe Faculty of Medicine, University of Oulu, for publicdiscussion in the Auditorium 12 of the University Hospitalof Oulu, on October 12th, 2001, at 12 noon.

OULUN YLIOPISTO, OULU 2001

Copyright © 2001University of Oulu, 2001

Manuscript received 29 August 2001Manuscript accepted 3 September 2001

Communicated byDocent Annika LaitinenDocent Aaro Miettinen

ISBN 951-42-6467-3 (URL: http://herkules.oulu.fi/isbn9514264673/)

ALSO AVAILABLE IN PRINTED FORMATISBN 951-42-6466-5ISSN 0355-3221 (URL: http://herkules.oulu.fi/issn03553221/)

OULU UNIVERSITY PRESSOULU 2001

Paananen, Reija, Characterization of surfactant proteins in porcine Eustachian tube Department of Paediatrics, University of Oulu, P.O.Box 5000, FIN-90014 University of Oulu,Finland, Biocenter Oulu, University of Oulu, P.O.Box 5000, FIN-90014 University of Oulu, Finland 2001Oulu, Finland(Manuscript received 29 August 2001)

Abstract

The Eustachian tube (ET) connects the upper respiratory tract and the middle ear. It equilibrates thepressure in the middle ear and prevents the harmful impact of the airways. Middle ear infection, orotitis media, is one of the most common illnesses in childhood and affects nearly all children at leastonce. ET dysfunction is considered to be a major pathogenic factor in the development of otitis media.

Surfactant proteins A (SP-A) and D (SP-D) have an important role in the pulmonary host defencesystem, and interactions with bacteria that also cause middle ear infections raised a question about theexpression of surfactant proteins in ET. The local defence system could be significant in preventingear infections.

The purpose of the study was to increase our basic knowledge of the putative ET surfactantsystem. ET and lung epithelia are both of endodermal origin, and ET epithelium with its mucociliarysystem resembles that of the lower airways. The aim was to determine whether SP-A, SP-B and SP-D are expressed in ET. The expressions were characterized with reverse transcriptase polymerasechain reaction (RT-PCR), Northern hybridizations and in situ hybridizations. The surfactant proteinswere localized using immunohistochemistry and immunoelectron microscopy. The proteins werecharacterized with Western analyses, and the properties of the ET surfactant were evaluated usingelectrospray ionization mass spectrometry and a pulsating bubble surfactometer.

SP-A, SP-B and SP-D were found to be expressed in porcine ET epithelium, and the cDNAsequences were homologous to those detected in lung tissue. The proteins were localized to specificET epithelial cells, and their sizes were characteristic of lung SP-A, SP-B and SP-D. ET lavage fluidcontained the surfactant proteins and phospholipid. However, the phospholipid molecular species andsurface tension measurements showed the structure and function of the surfactant in ET to be differentfrom lung surfactant, suggesting that a very low surface tension is not a critical determinant of ETsurfactant function.

As a conclusion, surfactant proteins in ET are likely to be involved in the local host defence systemand may contribute to the mucociliary function of the tube.

Keywords: gene expression, surfactant protein, Eustachian tube, phospholipid, surfacetension, middle ear

Acknowledgements

This work was carried out at the Department of Paediatrics and Biocenter Oulu, University of Oulu, during the years 1998-2001. I wish to express my deepest gratitude to my supervisor Professor Mikko Hallman for providing me the opportunity to do research in his group. His never-failing optimism and energy, his enthusiastic attitude towards research and encouragement have been essential for the accomplishment of this thesis.

I am grateful to Docents Annika Laitinen and Aaro Miettinen for their careful review and constructive comments on the manuscript. I also wish to thank Sirkka-Liisa Leinonen for the revision of the language of this thesis.

I would like to express my gratitude for fruitful collaboration to my co-authors, Virpi Glumoff, Ph.D., for her contribution and support during this work, Docent Raija Sormunen for her enormous knowhow in electron microscopy, Professor Anthony D. Postle, Wim Voorhout, Ph.D., Martin van Eijk, M.Sc., Graeme Clark, Ph.D., and Heike Dombrowsky, Ph.D.

I wish to thank all the people in our group for the warm atmosphere. I owe my special thanks to Ritva Haataja for her advice, encouragement and companionship especially during the last steps of this thesis. Sanna Eilola, Kirsi, Harju, Meri Lahti, Johan Löfgren, Marja Ojaniemi, Samuli Rounioja, Mika Rämet, Outi Väyrynen and our skilful technical assistants Maarit Hännikäinen, Mirkka Parviainen and especially Elsi Jokelainen, who made hundreds of excellent paraffin sections and participated in the Pouttu team, are also warmly acknowledged. I further wish to express my gratitude to Marjatta Paloheimo for her helpful attitude and for keeping everything in control.

I am grateful to my former workmates in the Departments of Biochemistry and Anatomy at the time when I was taking my first steps in the field of science, and to Professor Juha Peltonen for his kind references.

I wish to express my gratitude to Seija Seljänperä, Jorma Pudas and OYS cardiac surgeons for providing me the research material, and Sirpa Kellokumpu, Anna-Liisa Oikarainen, Tuomo Glumoff, Hong Min Tu, Marja Liisanantti and Emma Pirilä for their help and technical assistance during this work.

I wish to thank my friends for all the moments that turned my mind away from work and brought joy to my life. The meetings with our circle of woman friends have been of

special value. I would like to thank my two dogs “Rino” and “Jazi” for taking care of my mental and physical health and my friends in the OKK agility team.

I wish to express my love and respect to my parents Marjatta and Kari for always encouraging me and being willing to listen to my biochemical problems and to my sister Silja and her family as well as my brothers Jussi and Anssi.

Above all, I owe my warmest gratitude and love to my husband Kimmo for his love, understanding and patience.

This research was financially supported by Biocenter Oulu, The Academy of Finland and The Alma and K.A. Snellman foundation, Oulu, Finland.

Oulu, September 2001 Reija Paananen

Abbreviations

BAL(F) bronchoalveolar lavage (fluid) bp base pair(s) BSA bovine serum albumin cDNA complementary deoxyribonucleic acid CRD carbohydrate recognition domain C-terminal carboxy-terminal DPPC dipalmitoylated phosphatidylcholine, PC16:0/16:0 EM electron microscopy ET Eustachian tube ETL(F) Eustachian tube lavage (fluid) IEM immunoelectron microscopy IHC immunohistochemistry ISH in situ hybridization Ig immunoglobulin kb kilobase(s) kDa kilodalton(s) LPS lipopolysaccharide MBP mannose-binding protein mRNA messenger ribonucleic acid N-terminal amino-terminal PAGE polyacrylamide gel electrophoresis PBS phosphate-buffered saline PC phosphatidylcholine PE phosphatidylethanolamine PG phosphatidylglycerol PI phosphatidylinositol PS phosphatidylserine RDS respiratory distress syndrome ROM recurrent otitis media RSV respiratory syncytial virus RT-PCR reverse transcriptase polymerase chain reaction

SDS sodium dodecyl sulphate SP surfactant protein

List of original publications

This thesis is based on the following articles, which are referred to in the text by Roman numerals:

I Paananen R, Glumoff V & Hallman M (1999) Surfactant protein A and D expression in the porcine Eustachian tube. FEBS letters 452: 141-144.

II Paananen R, Glumoff V, Sormunen R, Voorhout W & Hallman M (2001) Expression and localization of lung surfactant protein B in Eustachian tube epithelium. Am J Physiol Lung Cell Mol Physiol 280: L214-L220.

III Paananen R, Sormunen R, Glumoff V, van Eijk M & Hallman M (2001) Surfactant proteins A and D in Eustachian tube epithelium. Am J Physiol Lung Cell Mol Physiol 281: L660-L667.

IV Paananen R, Postle AD, Clark G, Glumoff V & Hallman M (2001) Eustachian tube surfactant is different from alveolar surfactant. Determination of phospholipid composition of porcine Eustachian tube lavage fluid. Submitted.

Contents

Abstract Acknowledgements Abbreviations List of original publications 1 Introduction................................................................................................................... 13 2 Review of the literature ................................................................................................. 15

2.1 Pulmonary surfactant ............................................................................................. 15 2.1.1 Surfactant proteins (SPs)................................................................................. 15 2.1.2 Phospholipids in pulmonary surfactant ........................................................... 16 2.1.3 Airway surfactant ............................................................................................ 16 2.1.4 Non-mammalian surfactant ............................................................................. 17

2.2 Collectins ............................................................................................................... 17 2.2.1 Collectins and innate immunity....................................................................... 19

2.3 Surfactant Protein A (SP-A) ................................................................................... 19 2.3.1 SP-A genes ...................................................................................................... 19 2.3.2 SP-A expression and subcellular localization.................................................. 19 2.3.3 SP-A protein.................................................................................................... 20 2.3.4 SP-A secretion................................................................................................. 21 2.3.5 Functions of SP-A........................................................................................... 21

2.3.5.1 Interaction of SP-A with lipids................................................................. 22 2.3.5.2 Interaction of SP-A with micro-organisms............................................... 22

2.3.6 SP-A receptors................................................................................................. 23 2.3.7 SP-A-deficient mice ........................................................................................ 24 2.3.8 SP-A in lung diseases ...................................................................................... 26

2.4 Surfactant Protein D (SP-D)................................................................................... 26 2.4.1 SP-D gene ....................................................................................................... 26 2.4.2 SP-D expression and subcellular localization ................................................. 27 2.4.3 SP-D protein.................................................................................................... 27 2.4.4 Functions of SP-D........................................................................................... 28

2.4.4.1 Interaction of SP-D with lipids................................................................. 28 2.4.4.2 Interaction of SP-D with micro-organisms............................................... 28

2.4.5 SP-D receptors ................................................................................................ 29 2.4.6 SP-D-deficient mice ........................................................................................ 29 2.4.7 SP-D in lung diseases...................................................................................... 30

2.5 Surfactant Protein B (SP-B) ................................................................................... 30 2.5.1 SP-B gene........................................................................................................ 30 2.5.2 SP-B expression and subcellular localization.................................................. 31 2.5.3 SP-B protein.................................................................................................... 31 2.5.4 Functions of SP-B related to diseases ............................................................. 32

2.6 Surfactant Protein C (SP-C) ................................................................................... 33 2.7 Eustachian tube (ET).............................................................................................. 33

2.7.1 Cells in Eustachian tube epithelium ................................................................ 35 2.7.2 Eustachian tube and otitis media ..................................................................... 36 2.7.3 Pathogens involved in middle ear infections ................................................... 37 2.7.4 Surfactant in Eustachian tube.......................................................................... 38

3 Outlines of the present study......................................................................................... 39 4 Materials and methods .................................................................................................. 40

4.1 Eustachian tube preparations and bronchoalveolar lavage ..................................... 40 4.1.1 Preparation of total RNA (I, II) ....................................................................... 40 4.1.2 Preparation of samples for light microscopic and protein studies (II, III)....... 40

4.1.2.1 Preparation of aggregate fractions (II, IV) ............................................... 41 4.2 SP expression (I, II, III).......................................................................................... 41

4.2.1 Cloning of cDNA fragments for ET SP-A, SP-D and SP-B (I, II) .................. 41 4.2.2 Northern analysis (I, II)................................................................................... 41 4.2.3 In situ hybridization (II, III) ............................................................................ 42

4.3 Microscopy studies (I, II, III) ................................................................................. 43 4.3.1 Immunohistochemistry (I, II, III) .................................................................... 43 4.3.2 Electron microscopy (II, III) ........................................................................... 44 4.3.3 Immunoelectron microscopy (II, III)............................................................... 44

4.4 Western analysis (II, III)......................................................................................... 44 4.5 Analysis of phospholipid molecular species by electrospray ionization mass spectrometry (IV)......................................................................................................... 44 4.6 Surface activity measurements (IV) ....................................................................... 45

5 Results........................................................................................................................... 46 5.1 Expression of SP-A, SP-B and SP-D in ET (I, II, III)............................................ 46 5.2 Localization of SPs in ET (I, II, III) ....................................................................... 47 5.3 Immunoreactivities of ETLF (II, III)...................................................................... 48 5.4 Phospholipid composition of ETLF (IV) ............................................................... 49 5.5 Surface activity of ETLF (IV) ................................................................................ 50

6 Discussion ..................................................................................................................... 51 7 Conclusions................................................................................................................... 58 8 References..................................................................................................................... 59

1 Introduction

Pulmonary surfactant consists of lipids and proteins, and it reduces the surface tension in the air/liquid surfaces of alveoli. It is also involved in the pulmonary host defence system. Pulmonary surfactant contains specific proteins, of which SP-B, SP-C and SP-A enhance the surface activity of phospholipid. Collagenous glycoproteins, SP-A and SP-D, additionally have immunomodulatory functions. They bind to the surface of various micro-organisms and facilitate phagocytosis by macrophages.

Eustachian tube (ET) embryonally develops from the first pharyngeal pouch and connects the upper respiratory tract to the middle ear. ET protects the middle ear from excessive deviations of atmospheric pressure, serves as a clearance tract, and protects the middle ear against the invasion of microbes and other noxious agents from the airways. Chronic middle ear infections may result in hearing loss and delays in speech and language development. ET in children is structurally and functionally different from that in adults, which leads to impaired function of the tube and could explain the proneness of infants to middle ear infections. Although ET dysfunction is considered the principal pathogenic factor in the susceptibility to recurrent middle ear infections, the mechanisms and the potential genes involved are still unknown.

The existence of a surfactant-like substance in ET has been under discussion for decades. Also, experiments to treat middle ear infections with synthetic surfactants have been reported. At the time when this work was started, surfactant proteins were mainly considered lung-specific. A few reports on the expression of SP-A in intestinal tract had been published, and two studies suggested the presence of SP-A in human middle ear epithelium and effluent. Recent studies have shown that SP-D contributes to the host defence functions of many mucosal surfaces, and SP-A is essentially involved in the clearance of pathogens. SP-B has been regarded as lung-specific.

According to the current hypothesis, the local surfactant system of ET is of major functional significance. The ET epithelium closely resembles the airway epithelium, but there are major differences in the functions of these surfaces. Unlike in the lower respiratory tract, closure takes place in the ET. The lumen of the ET is also wider than that of airspaces and small airways, and no need for such a potent surface tension reducing agent may therefore be evident.

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This study demonstrates that SP-A, SP-B and SP-D are expressed in porcine ET. Additionally, these proteins were localized within the epithelium. The phospholipid structure and surface properties of ET lavage fluid were determined and compared to those isolated from bronchoalveolar lavage fluid.

This study yielded novel knowledge of the ET surfactant system that may prove important in developing more effective treatment or prevention of middle ear infections.

2 Review of the literature

2.1 Pulmonary surfactant

Pulmonary surfactant is a complex of lipids and proteins that lines the interior of the lung. Pulmonary surfactant prevents alveolar collapse by reducing the surface tension across the air/liquid interface of the alveoli (Clements 1977). A lack of surfactant causes a disturbance of alveolar gas exchange. This can be seen in premature infants suffering from respiratory distress syndrome (RDS), a major cause of neonatal death (Farrell & Avery 1975). Surfactant is also involved in several defence mechanisms in the lung. In small airways, surfactant acts to maintain patency (Enhorning 1977). It also prevents the increase in airway resistance in allergen-challenged animals (Liu et al. 1996) and enhances mucociliary clearance of harmful contaminants (Im Hof et al. 1997, De Sanctis et al. 1994). The pulmonary surfactant complex consists of approximately 90 % lipid and 10 % protein, including four surfactant proteins (SP-A, B, C and D).

2.1.1 Surfactant proteins (SPs)

The most abundant surfactant protein, SP-A, was first identified by King and Clements in 1972 (King & Clements 1972). Despite the early discovery and intensive investigations, the function of SP-A remains perhaps the most controversial of the four surfactant proteins. Most studies have focused on the role of SP-A in surfactant structure and function, but today, SP-A is also regarded as important in pulmonary host defence. Two hydrophobic surfactant proteins, SP-B and SP-C, are critical for the adsorption and spreading of the surfactant film at the air/liquid interface. SP-B is required for the normal processing of the surfactant components in alveolar type II epithelial cells, and SP-C is a highly hydrophobic alveolar type II epithelial cell-specific protein that contributes to the formation and maintenance of the surface-active monolayer (Batenburg & Haagsman 1998). SP-A and SP-D, being C-type lectins, participate in surfactant homeostasis and host defence.

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SP-A, SP-B and SP-D are expressed primarily in alveolar type II epithelial cells and bronchiolar epithelial (Clara) cells, whereas SP-C is detected only in alveolar type II epithelial cells (Phelps & Floros 1988, Kalina et al. 1992, Khoor et al. 1993, Wong et al. 1996).

2.1.2 Phospholipids in pulmonary surfactant

The overall lipid and phospholipid compositions of mammalian surfactants appear to be very similar in different species. In surface-active material, about 80% of lipids are phospholipids, whereas cholesterol comprises the largest amount of neutral lipid. Phospholipids are crucial for the physiological function of lung surfactant. Phosphatidylcholine (PC) is the main phospholipid in surfactant, comprising approximately 70-80% of phospholipids, and about half of it is dipalmitoylated phosphatidylcholine (DPPC or 16:0/16:0PC). DPPC is the major surface-active component. The disaturated nature of DPPC enables surfactant to withstand very high surface pressure, and this is thought to prevent the collapse of small alveoli and conducting airways (Enhorning et al. 1995). Although DPPC is an unusual molecular species, it is not specific for PC in the surfactant compartments. DPPC can be detected in other tissues as well. However, the percentage of DPPC in lung surfactant PCs is uniquely high compared to other tissues. The other major molecular species of lung surfactant phospholipids include palmitoyl-oleoyl (16:0/18:1), palmitoyl-palmitoleoyl (16:0/16:1) and palmitoyl-linoleoyl (16:0/18:2) (Akino 1992).

The acidic phospholipids, phosphatidylglycerol (PG) and phosphatidylinositol (PI) comprise approximately 8-15% of the total surfactant phospholipid pool in most species. The PG concentration in surfactant is very high compared to other mammalian tissues, where only very small amounts of PG can be detected (Hallman & Gluck 1976). However, PG is not essential for the surface activity of pulmonary surfactant. It can be replaced by PI without impairing the surfactant function (Hallman & Gluck 1976, Beppu et al. 1983).

The remaining phospholipids in surfactant are phosphatidylethanolamine (PE) and phosphatidylserine (PS), which are present in rather small amounts (Akino 1992).

Cholesterol is the major component of neutral lipids in pulmonary surfactant. Small amounts of monoacylglycerol, diacylglycerol and triacylglycerol are present in most species (Yu et al. 1983).

2.1.3 Airway surfactant

Conductive airway surfactant is similar to alveolar surfactant in terms of phospholipid classes and PC molecular species (Bernhard et al. 1997). However, the surface tension function of conductive airway surfactant is impaired in comparison with alveolar surfactant (Bernhard et al. 1997). This conductive airway surfactant could be derived from the overflow of alveolar material or from surface-inactive alveolar surfactant, since the PL and PC compositions of the underlying airway epithelium are remarkably

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different. Although Clara cells of airway epithelium express the surfactant proteins A, B and D, no lamellar bodies, which are the storage organelles of surfactant, can be detected in these cells (Voorhout et al. 1992a). The phospholipid classes and PC molecular species compositions of airway mucosa are similar to those in gastric mucosa (Bernhard et al. 1995), where SP-A and SP-D expressions have also been detected (Chailley-Heu et al. 1997).

2.1.4 Non-mammalian surfactant

The pulmonary surfactant system, including both lipids and SP-A, is present in the lungs of air-breathing representatives of all vertebrates studied (Sullivan et al. 1998). In fact, it appears that the surfactant function related to air breathing evolved at least 400 million years ago in the lungs of primitive air-breathing fish. Thus, the essential function is highly conserved (Sullivan et al. 1998). Surfactant has also been found in the swim bladders of modern teleost fish (Daniels et al. 1994, Smits et al. 1994, Prem et al. 2000). As the swim bladder is not required for air breathing, this finding suggests that surfactant is also requisite for an inflating/deflating organ with an air/liquid interface. The surfactant discovered in non-mammalian vertebrates is generally less disaturated, and its surface activity hence tends to be relatively low. These considerations have led to the suggestion that surfactant has other functions in non-mammalian vertebrates, such as acting as an anti-adherent (Veldhuizen et al. 1998).

2.2 Collectins

The surfactant proteins SP-A and SP-D are hydrophilic collagenous glycoproteins (Sastry & Ezekowitz 1993). Together with the serum mannose-binding protein (MBP) (Drickamer et al. 1986), serum bovine conglutinin (Lee et al. 1991), serum bovine collectin-43 (Lim et al. 1994a) and the recently described human liver collectin CL-L1 (Ohtani et al. 1999), they belong to the family of C-type lectins (collectins). They consist of a short amino-terminal region, a long collagen-like domain, a neck region and a carbohydrate recognition domain (CRD). The amino-terminal disulfide-rich domain of 7-25 amino acids contributes to interchain covalent interactions, and the collagen-like domain forms a triple helix of 20-46 nm. The neck region is a short trimeric coiled coil, which bridges the collagenous domain to the CRD. The basic structural unit of each collectin is a trimer based on a collagen-like triple helix (Holmskov et al. 1994) (Fig. 1a).

All collectins form multimers, although the degree of multimerization varies. SP-A and MBP form a bouquet-like octadecamer comprising six trimeric subunits (Fig. 1b). MBP also forms oligomers of two, three, four and five trimeric subunits (Lipscombe et al. 1995). With their more extended collagen-like domains, conglutinin and SP-D form dodecamers comprising four trimeric subunits (Fig. 1c), although both larger (Crouch et al. 1994a) and smaller forms of SP-D have been identified (Lu et al. 1993). Multimerization seems to be an important determinant in the interaction of collectins with immune cells and pathogens (Brown-Augsburger et al. 1996).

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Lung collectins specifically recognize and interact with a wide range of micro-organisms and show specific interactions with leukocytes (For a review, see Crouch & Wright 2001). They also modulate the function of phagocytic cells in vitro and in vivo. According to the current evidence, the main role of collectins is to interact directly with carbohydrate on the surface of microbial pathogens, thereby initiating different effector mechanisms. They also interact with and modulate the cellular effects of inhaled pollens and other complex organic antigens (Malhotra et al. 1993, Wang et al. 1996).

a)

N-terminal collagenous α-helical C-type noncollagenous region neck lectin region region domains

b) c)

Fig. 1. a) Schematic structure of a collectin trimer. b) Structure of SP-A. Six trimers oligomerize and form a structural SP-A unit. c) Structure of SP-D. An oligomer of four trimers. (Modified from Wright 1997).

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2.2.1 Collectins and innate immunity

Collectins have been implicated in the innate, non-antibody-mediated immune defence system (For recent reviews, see Holmskov 2000, Crouch & Wright 2001). They have several features that may be expected of an ideal molecule that plays a role in first-line non-clonal immunity. Collectins bind to carbohydrates on various micro-organisms and to specific receptors on phagocytic cells, thus accelerating microbial clearance. Collectins can aggregate pathogens, neutralize viruses, attract and activate phagocytes and promote phagocytosis. Local sequence differences in the CRDs of collectins dictate the repertoire of ligand recognition (Epstein et al. 1996). Individual collectins and their specific structural domains are likely to have particular roles in host defence (Hartshorn et al. 2000a, Hartshorn et al. 2000b).

Mannose-binding protein (MBP) has been a target of profound studies. It is predominantly a serum protein, but it has also been detected in amniotic fluid (Malhotra et al. 1994a), nasal secretions, middle ear fluid (Garred et al. 1993) and inflamed sites, such as rheumatic joint fluid (Malhotra et al. 1995). It has been associated with a variety of diseases, including various infectious diseases as well as autoimmune diseases (Holmskov 2000). For example, children deficient in MBPs are more susceptible to recurrent infections (Super et al. 1989). Recently, it was shown that specific SP-A haplotypes occur at an increased frequency in children susceptible to recurrent otitis media (ROM) (Rämet et al. 2001).

2.3 Surfactant Protein A (SP-A)

2.3.1 SP-A genes

Primates have two genes coding SP-A; SP-A1 and SP-A2, and a pseudogene (Korfhagen et al. 1991). The human SP-A genes (Fisher et al. 1987) as well as two other collectins, SP-D and MBP (Kolble & Reid 1993), are located on the long arm of chromosome 10, spanning from 10q21 to 10q24. The SP-A gene is approximately 4.5 kb in length, consisting of 5 exons and 4 introns (White et al. 1985). Other species, with the exception of primates, are known to have just a single gene coding SP-A. The significance of these two very similar genes is not known, but the patterns of their expression could differ, SP-A2 expression being mainly detected in the upper airways (Saitoh et al. 1998). These genes are also differentially regulated during development (Scavo et al. 1998).

2.3.2 SP-A expression and subcellular localization

Human SP-A mRNA is 2.2 kb in length (Ballard et al. 1986). In other species, mRNAs ranging from 1.0 kb to 3.0 kb in size have been detected (Boggaram & Mendelson 1988, Fisher et al. 1988). SP-A is expressed and synthesized mainly in alveolar type II

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epithelial cells and non-ciliated epithelial (Clara) cells (Phelps & Floros 1988, Voorhout et al. 1992b). SP-A expression is additionally characterized in tracheal and bronchial epithelium and in submucosal glands of the lower airways (Endo & Oka 1991, Khoor et al. 1993). More specifically, SP-A mRNA is detected in the mucosal folds where tracheal glands arise in human fetal lung. Nonmucous cells of tracheal glands show SP-A expression as they become differentiated (Hawgood 1992). SP-A2 mRNA has been detected in serous cells of submucosal glands of adult human conducting airways (Khubchandani & Snyder 2001). Recently, SP-A expression has been detected in middle ear epithelium and in submucosal glands in paranasal sinuses (Dutton et al. 1999). SP-A mRNA has further been reported to occur in rat intestinal epithelium (Rubio et al. 1995) and in a phospholipid-rich layer in rat and human colon (Eliakim et al. 1997) as well as in human prostate and thymus (Lu 1997). The different expression patterns of the SP-A1 and SP-A2 genes have been considered, and SP-A2 is probably the major gene expressed in airways and other extra-alveolar sites (Dutton et al. 1999).

SP-A protein has been detected in human seminal fluid (Khubchandani & Snyder 2001). Immunoreactivity for SP-A has been found in synovial intima and mesothelial cells of pleura, pericardium and peritoneum (Dobbie 1996). The suggested function for SP-A in these tissues is to aid in lubrication, to reduce surface tension and to prevent the development of adhesions. SP-A as a part of the host defence system in the ductal epithelium of lacrimal and salivary glands has been the explanation for the immunoreactivity found in these tissues (Dobbie 1996). SP-A protein, but not mRNA, has been detected in macrophages, probably due to the uptake of SP-A (Khoor et al. 1993).

SP-A protein is seen in lamellar bodies of type II cells, but at a relatively low level. Mainly SP-A is found in tubular myelin structures in the alveolar lining and in immature and mature secretory organelles in the Clara cells of rat lung (Voorhout et al. 1991a, Voorhout et al. 1992b).

2.3.3 SP-A protein

The human SP-A preprotein consists of 255 amino acids, and after signal peptide cleavage, the mature form consists of 228 amino acids, coding a 28-kDa protein. In intracellular processing of SP-A, asparagines are glycosylated (McCormack et al. 1994), i.e. complex sugar chains are added, and prolines within the collagen-like region are hydroxylated (Weaver & Whitsett 1991). The biological activities of rat SP-A are critically dependent on intact disulfide bonds (Kuroki et al. 1988a). The glycosylation of SP-A is species-specific, resulting in 28-36-kDa proteins, and glycosylated SP-A is thought to be an immunologically more active form of SP-A (van Iwaarden et al. 1992). Immunoreactive forms of pulmonary SP-A are known to display notable heterogeneity. This is thought to be the result of differential glycosylation and acylation processes occurring at the post-translational level (Weaver et al. 1985, Whitsett et al. 1985) and of the presence of non-reducible multimeric forms (Kuroki et al. 1988a).

Subunits of SP-A form trimers by helical folding of their collagen-like regions. These are further oligomerized into a structure of six SP-A trimers, which resembles a bouquet of tulips (Voss et al. 1988) (Fig. 1b). Hattori and his co-workers (Hattori et al. 1996)

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found two distinct oligomeric structures of human SP-A, which seemed to exhibit different capacities to interact with alveolar type II epithelial cells. Recently, there has been a report of several different oligomeric states of SP-A (Hickling et al. 1998). Ligand-binding affinity is greatly enhanced by the multimeric organization of the protein, which allows for simultaneous interaction with several sites on multivalent ligands or membranes (Damodarasamy et al. 2000).

Different parts of the SP-A molecule have different functions. CRD is known to affect lipid binding and pH-dependent lipid aggregation (McCormack et al. 1997a, McCormack et al. 1997b). CRD also interacts with carbohydrates on the surface of micro-organisms. The N-terminal domain is essential for lipid aggregation and contains the structural elements required for the oligomeric assembly of SP-A (Elhalwagi et al. 1997). N-terminal interchain bonds and the C-terminal half of the collagen-like region for SP-A modulate the surfactant uptake by type II cells (McCormack et al. 1999). The trimeric subunit of SP-A is stabilized by noncovalent interactions between the coiled coil α-helices of the neck region and by the collagenous triple helix. The neck region thus determines the spatial orientation of the CRDs. The neck domain may also be involved in the process of the SP-A-mediated uptake of phospholipids (Sano et al. 1998).

2.3.4 SP-A secretion

Previously, the lamellar body of the type II cell was considered to contain all the lipid and protein components of surfactant. Quantitatively, however, SP-A only accounts for 1% of the total protein content within intracellular lamellar bodies, and this amount is insufficient for proper arrangement or tubular myelin structure in the alveolus. With exogenous SP-A addition, maximal surface activity and structural rearrangement of lamellar bodies into organized multilamellate forms was achieved in cultured human foetal lung cells (Froh et al. 1990). This observation gave rise to studies which confirmed that only 10% of secreted SP-A is released within lamellar bodies, most of it being secreted via alternative routes (Froh et al. 1993). In Clara cells of rat lung, SP-A is detected in secretory granules, which are thought to be lipid-poor (Voorhout et al. 1991a, Voorhout et al. 1992b). Actual secretion of SP-A from Clara cells has not been documented thus far.

2.3.5 Functions of SP-A

SP-A binds to alveolar type II epithelial cells and immune cells, particularly alveolar macrophages (van Iwaarden et al. 1990). SP-A also contributes to the formation and maintenance of the tubular myelin structure, the typical form of extracellular alveolar surfactant (Suzuki et al. 1989). Although tubular myelin is present in all mammals, the functional significance of this form remains unknown.

The proposed functions of SP-A in vitro include enhancement of the surface activity of phospholipids (Hawgood et al. 1987) and maintenance of homeostasis between the extra- and intracellular surfactant pools (Dobbs et al. 1987, Rice et al. 1987). SP-A can

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modulate the uptake and secretion of surfactant from isolated alveolar type II epithelial cells by a receptor-mediated mechanism (Wright 1990). SP-A both increases the uptake of DPPC into type II cells and decreases the regulated secretion of lamellar bodies from these cells (Kuroki et al. 1988b).

Today, however, the role of SP-A in non-antibody-mediated defence against micro-organisms is considered evident and increasingly important, since no obvious homeostatic defect in gas exchange or lung mechanics was detected in the developed SP-A knockout mouse (Korfhagen et al. 1996).

2.3.5.1 Interaction of SP-A with lipids

SP-A interacts with lipids and specifically binds to DPPC, the major surfactant phospholipid, in a calcium-dependent manner (Kuroki & Akino 1991). SP-A acts as an inhibitor of phospholipid secretion from rat alveolar type II epithelial cells (Dobbs et al. 1987, Rice et al. 1987) and binds to the surface of these cells (Kuroki et al. 1988c, Wright et al. 1989). SP-A also enhances the uptake of lipids by type II cells (Wright et al. 1987) and improves the SP-B-mediated surface tension-reducing properties of surfactant lipids (Veldhuizen et al. 2000). SP-A additionally binds to the lipid A domain of gram-negative lipopolysaccharide (LPS) (van Iwaarden et al. 1994, Kalina et al. 1995) and to several glycolipids and neutral glycosphingolipids (Childs et al. 1992, Kuroki et al. 1992a, Momoeda et al. 1996).

2.3.5.2 Interaction of SP-A with micro-organisms

SP-A interacts with a variety of micro-organisms and enhances the uptake of some by inflammatory cells. Table 1 summarizes the micro-organisms that SP-A has been shown to interact with.

The first study on this topic showed that human SP-A enhances the uptake of Staphylococcus aureus by rat alveolar macrophages (van Iwaarden et al. 1990). Today, SP-A is known to enhance the uptake of various other bacteria, such as Haemophilus influenzae, type A (McNeely & Coonrod 1994), Pseudomonas aeruginosa (Manz-Keinke et al. 1992), Streptococcus pneumoniae and group A Streptococcus (Tino & Wright 1996). SP-A can interact with bacterial cell wall constituents, such as the di-mannose repeating unit associated with some capsular polysaccharides. It can also interact with the lipid A domain of Escherichia coli (E. coli) LPS (van Iwaarden et al. 1994, Kalina et al. 1995) or the outer membrane protein of Haemophilus influenzae, type A (McNeely & Coonrod 1994).

SP-A also interacts with various respiratory viruses. It can inhibit the infectivity and hemagglutination of influenza A virus in vitro (Benne et al. 1995, Hartshorn et al. 1997). SP-A promotes the uptake of herpes simplex virus by rat alveolar macrophages (van Iwaarden et al. 1991) and can lower the infectivity of respiratory syncytial virus (RSV) (Ghildyal et al. 1999). Additionally, SP-A interacts with cell wall glycoconjugates of fungi, for example Pneumocystis carinii, through its CRD (McCormack et al. 1997b).

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SP-A enhances the uptake of pathogens by at least two mechanisms, directly as an opsonin, which means that SP-A binds to particulate antigens and induces their phagocytosis by macrophages and neutrophils, and indirectly as an activation ligand. As an opsonin, SP-A coats micro-organisms and facilitates their uptake by direct interaction with phagocytic cells. SP-A binds directly to the surface of some strains of bacteria, including Staphylococcus aureus, Streptococcus pneumoniae (McNeely & Coonrod 1993), Haemophilus influenzae (McNeely & Coonrod 1994), Klebsiella pneumoniae (Kabha et al. 1997) and Mycobacterium tuberculosis (Pasula et al. 1997).

SP-A can also enhance phagocytosis indirectly as an activation ligand, which is a factor that enhances the uptake of particles opsonized with another factor. SP-A has been shown to enhance the uptake of particles opsonized with IgG (Tenner et al. 1989). SP-A may additionally instruct immune cells by recruiting phagocytic cells at sites of inflammation and immune action. SP-A can act as chemoattractant for neutrophils and monocytes (Wright & Youmans 1993). Purified SP-A, as well as SP-D, often contain endotoxin. Thus, in many preparations, endotoxin contamination may be due to the interaction of proteins with bacteria or bacterial LPS.

SP-A appears to have three models proposed for cell binding: via a lectin-mediated event to monocytes and macrophages (Wintergerst et al. 1989, Manz-Keinke et al. 1991), by a macrophage cell-surface lectin that binds the N-linked carbohydrate on the CRD (Gaynor et al. 1995), and to alveolar macrophages via collagenous stalks (Wright & Youmans 1993).

SP-A not only participates in lung defence against pathogens but also modulates inflammatory and allergic responses. SP-A can bind to a number of different pollen grains and to specific water-soluble glycoproteins from these pollens in vitro (Malhotra et al. 1993). SP-A also plays an important role in suppressing immune responses to other inhaled allergens, such as Aspergillus fumigatus and mite in vitro (Wang et al. 1996, Madan et al. 1997a, Madan et al. 1997b, Wang et al. 1998) and protects mice against allergic reactions induced by Aspergillus fumigatus (Madan et al. 2001).

2.3.6 SP-A receptors

Both rat alveolar macrophages and type II epithelial cells express receptors for SP-A. SP-A binds to both cell types with high but different affinities. This binding is saturable, specific and at least partly calcium-dependent (Kuroki et al. 1988c, Wright et al. 1989, Pison et al. 1992). It has been suggested that both the collagen-like domain and the CRD of SP-A may interact with cell surface receptors.

There has been evidence of several proteins as candidates for SP-A receptors on human macrophages, and two of the receptors are shared by C1q. The C1q-binding protein of 56 kDa additionally binds other collectins but not SP-D. This binding does not require calcium and appears to be mediated by the collagen-like domain of the proteins (Malhotra et al. 1990). A 126-kDa protein named C1qRp (Nepomuceno et al. 1997) binds C1q, mannose-binding lectin and SP-A. This receptor has been shown to be involved in the SP-A-mediated uptake of Staphylococcus aureus by monocytes (Geertsma et al. 1994).

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A 210-kDa protein from lung membranes has been purified with SP-A affinity chromatography (Chroneos et al. 1996). This receptor is found on type II epithelial cells and alveolar macrophages from rat lung. SP-A binding to this receptor is calcium-dependent and not based on carbohydrate recognition. These findings also support the hypothesis that multiple receptors may exist for the collectins on immune cells.

SP-A has recently been shown to bind to CD14, a known receptor for LPS, which is a component of the outer membrane of gram-negative bacteria that is responsible for sepsis and induction of inflammation (Sano et al. 1999). Among human and porcine alveolar type II epithelial cells, two different SP-A binding proteins were identified (Strayer et al. 1993, Wissel et al. 1996).

2.3.7 SP-A-deficient mice

The deletion of SP-A gene expression apparently does not affect lung stability (Korfhagen et al. 1996). No abnormalities in histology, lung wet weight, lung volume or compliance could be detected in SP-A-deficient mice. These mice completely lack tubular myelin. The surface activity of surfactant, however, was not different from that of the controls. The surfactant from SP-A (-/-) mice is different in structure, density, and several other properties from normal surfactant, but these differences do not result in abnormal lung function (Ikegami et al. 1998, Korfhagen et al. 1998). These studies suggest that the effect of SP-A on biophysical properties and surfactant metabolism could have been overestimated on the basis of in vitro tests, or some compensatory adaptations may occur in vivo.

The importance of SP-A in host defence has been revealed in vivo. SP-A knockout mice have reduced defence against several lung pathogens, including group B Streptococci (LeVine et al. 1997, LeVine et al. 1999a), Pseudomonas aeruginosa (LeVine et al. 1998), Haemophilus influenzae (LeVine et al. 2000), RSV (LeVine et al. 1999b), and adenoviral infection (Harrod et al. 1999). SP-A-deficient mice showed enhanced susceptibility to infection, which was detected as increased colony counts in lung postinfection and as increased dissemination of group B Streptococcus to the spleen. Exogenous SP-A administered with the pathogens was able to restore the impaired host defence of these SP-A (-/-) mice. The number of bacteria associated with alveolar macrophages was decreased in the SP-A-deficient mice. With these micro-organisms, SP-A acts as an opsonin and enhances the ingestion of pathogens by interacting with macrophages (Kabha et al. 1997, Hickman-Davis et al. 1998, Koziel et al. 1998). Studies with SP-A-deficient mice suggest that SP-A modulates the clearance of pathogens via macrophages and demonstrates the important host defence role of SP-A.

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Table 1. SP-A and SP-D interactions with different micro-organisms.

Micro-organisms SP-A References SP-D References Bacteria Gram-negative (LPS)

Escherichia coli J5 X van Iwaarden et al. 1994, Pikaar et al. 1995, Stamme & Wright 1999

X Kuan et al. 1992, Pikaar et al. 1995, Hartshorn et al. 1998

Escherichia coli K12 X Manz-Keinke et al. 1992 Pseudomonas aeruginosa X Manz-Keinke et al. 1992, Tino &

Wright 1996, LeVine et al. 1998 X Lim et al. 1994b,

Restrepo et al. 1999 Haemophilus influenzae X McNeely & Coonrod 1994, Pikaar

et al. 1995, LeVine et al. 2000

Klebsiella pneumoniae X Kabha et al. 1997 X Lim et al. 1994b, Ofek et al. 2001

Gram-positive Group B Streptococcus X LeVine et al. 1997, LeVine et al.

1999a

Group A Streptococcus X Tino & Wright 1996 Streptococcus pneumoniae X Tino & Wright 1996, Hartshorn et

al. 1998 X Hartshorn et al. 1998

Staphylococcus aureus X van Iwaarden et al. 1990, Manz-Keinke et al. 1992, McNeely & Coonrod 1993, Geertsma et al. 1994, Hartshorn et al. 1998

X Hartshorn et al. 1998

Fungi Pneumocystis carinii X Zimmerman et al. 1992, Williams

et al. 1996, McCormack et al. 1997b

X Limper et al. 1995, O'Riordan et al. 1995, Vuk-Pavlovic et al. 2001

Aspergillus fumigatus X Madan et al. 1997a, Madan et al. 1997b, Madan et al. 2001

X Madan et al. 1997a, Madan et al. 1997b, Allen et al. 1999, Madan et al. 2001

Yeasts Cryptococcus neoformans X Walenkamp et al. 1999 X Schelenz et al. 1995 Candida albicans X Rosseau et al. 1997, Rosseau et al.

1999 X van Rozendaal et al.

2000 Viruses

Influenza A virus X Malhotra et al. 1994b, Benne et al. 1995, Tino & Wright 1996, Benne et al. 1997

X Hartshorn et al. 1994, Hartshorn et al. 1996a, Hartshorn et al. 1997

Herpes simplex virus X van Iwaarden et al. 1991, van Iwaarden et al. 1992

Respiratory syncytial virus X Ghildyal et al. 1999, LeVine et al. 1999b, Barr et al. 2000, Hickling et al. 2000

X Hickling et al. 1999, LeVine & Whitsett 2001

Bacillus Calmette-Guérin X Weikert et al. 1997, Weikert et al. 2000

Others Mycoplasma pulmonis X Hickman-Davis et al. 1998,

Hickman-Davis et al. 1999

Mycobacterium tuberculosis

X Downing et al. 1995, Gaynor et al. 1995, Pasula et al. 1997, Pasula et al. 1999

X Ferguson et al. 1999

Particulate matter Pollen X Malhotra et al. 1993 Mite extract X Wang et al. 1996 X Wang et al. 1996

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2.3.8 SP-A in lung diseases

SP-A levels have been shown to be altered in a variety of pulmonary diseases (For a review, see Kuroki et al. 1998). SP-A levels may be altered secondarily by changes in steroids, growth factors or cytokines, the levels of which may vary in different pathologic conditions. However, it could also be speculated that suppressed levels of SP-A may result in increased susceptibility to disease. The two human SP-A genes are highly polymorphic, and there may be allele-specific differences in SP-A mRNA levels. This suggests that either the SP-A protein levels may be genetically determined or an allele affecting the amino acid sequence of SP-A may have functional consequences (Karinch et al. 1997, Floros & Kala 1998).

SP-A levels in amniotic fluid are useful indicators of lung maturity and predict the probability of RDS in premature infants (Hallman et al. 1988). The levels of SP-A in airway specimens are low in infants who develop chronic lung disease and increase during recovery from RDS (Chida et al. 1988, Hallman 1991). A decrease in SP-A levels has also been observed in the bronchoalveolar lavage fluid (BALF) of adult patients with acute RDS (ARDS) (Kuroki et al. 1998).

Elevated levels of SP-A in BAL fluids and sputum can be detected in pulmonary alveolar proteinosis (Singh et al. 1983, Masuda et al. 1991, Honda et al. 1993). In contrast, SP-A levels in BALF were lower than normal in patients with idiopathic pulmonary fibrosis (McCormack et al. 1991) and in both gram-positive and gram-negative bacterial pneumonia (Baughman et al. 1993). However, some controversy exists. There are also studies reporting increased SP-A concentrations in pulmonary bacterial infection (Phelps & Rose 1991, Hull et al. 1997). These conflicts may be due to the difficulties of quantifying SP-A or the use of different assay methods. The detected serum SP-A concentrations increase in these diseases (Kuroki et al. 1993, Honda et al. 1995a, Takahashi et al. 1995), but the levels measured from BAL fluids tend to be lower.

A reduced level of SP-A is seen in RSV infection (Kerr & Paton 1999). However, it is not known whether the reduced level of SP-A predisposes to infection or whether SP-A is consumed in RSV infection when SP-A acts as an opsonin.

2.4 Surfactant Protein D (SP-D)

2.4.1 SP-D gene

The mouse SP-D gene extends over 14 kb and consists of 8 exons. The positions of introns are conserved between the mouse and human SP-D genes (Lawson et al. 1999). The primary structures of SP-D have been characterized by cDNA cloning in human (Rust et al. 1991, Lu et al. 1992), rat (Shimizu et al. 1992), mouse (Motwani et al. 1995), cow (Lim et al. 1993) and pig (van Eijk et al. 2000).

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2.4.2 SP-D expression and subcellular localization

The main sites of SP-D synthesis and secretion include alveolar type II and non-ciliated airway epithelial cells, Clara cells (Voorhout et al. 1992b). SP-D expression is also detected in the epithelium of the conducting airways and in the tracheal and bronchial glands of the lower airways (Wong et al. 1996). Today, SP-D is increasingly recognized as a molecule involved in the host defence system of several organs, and expression has been detected with RT-PCR in many mucosal surfaces in human tissues, including kidney, brain and testis (Madsen et al. 2000).

SP-D protein has been localized with immunoelectron microscopy to amorphous granular material in airspaces and to rough endoplasmic reticulum in alveolar type II cells. The protein has not been detected in lamellar bodies (Crouch et al. 1991, Voorhout et al. 1992b), but it could be localized in the periphery of apical secretory granules of rat bronchiolar nonciliated cells (Crouch et al. 1992, Voorhout et al. 1992b). In addition, the apical surface of the Clara cells was highly labelled (Crouch et al. 1991). There are differences in the processing of SP-D between Clara cells and alveolar type II epithelial cells (Mason et al. 1998).

2.4.3 SP-D protein

SP-D is one of the largest molecules found in the innate immune system. Its X-shaped tertiary structure of four trimers is more than 100 nm long (Crouch et al. 1994b) (Fig. 1c). The SP-D monomer is a 43-kDa protein. Similarly to SP-A, SP-D consists of isoforms that differ in apparent molecular weight and isoelectric point (Crouch 1998). As for other collectins, the modifications of SP-D include cleavage of a signal peptide and partial hydroxylation of proline and lysine residues in the collagen-like region (Holmskov et al. 1995). SP-D monomer chains form subunits of homotrimers, which then oligomerize into cross-like tetramers and higher oligomers. SP-D is non-sedimentable at neutral pH (Persson et al. 1990). Dodecamer is the dominant form of native SP-D, but single subunits as well as multimers of up to eight dodecamers have been observed for rat SP-D (Crouch et al. 1994b). The dominant form of human SP-D is a 37- to 41-kDa polypeptide (Leth-Larsen et al. 1999), but a form of human SP-D with a reduced mass of 50 kDa has also been detected (Mason et al. 1998). This increase in molecular weight is due to O-linked glycosylation.

According to the recent studies, porcine SP-D has three structural differences compared to other species. One of these is an extra cystein in the collagenous region. This could lead to a different process of oligomerization and to a more stable heteromer. The other differences are the insertion of three amino acids and a potential extra N-glycosylation site in the CRD (van Eijk et al. 2000).

The CRD of SP-D is responsible for its lectin activity, and trimeric clusters of SP-D CRDs are required for high-affinity binding to multivalent ligands (Kishore et al. 1996, Hakansson et al. 1999).

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2.4.4 Functions of SP-D

SP-D does not bind to the surfactant complex or enhance surface activity. However, it binds directly to type II cell apical membranes (Herbein et al. 2000) and alveolar macrophages (Miyamura et al. 1994). It also binds to carbohydrate structures on the surfaces of a variety of pathogens, including bacteria (Kuan et al. 1992), viruses (Hartshorn et al. 1994), yeasts (Schelenz et al. 1995), fungi (Madan et al. 1997b) and inhaled pollens and other complex organic antigens (Wang et al. 1996). A summary of the micro-organisms with which SP-D interacts is presented in Table 1. The binding initiates several effector mechanisms, including the recruitment of inflammatory cells to destroy pathogens (Holmskov 1999) and the modulation of the immune cell and cytokine response (Borron et al. 1998). In addition to its role in antimicrobial defence, SP-D is involved in pulmonary surfactant homeostasis, even though it does not interact with the major surfactant phospholipids and is not associated with lamellar bodies or tubular myelin. Nor did it have a detectable effect on surfactant secretion from alveolar type II epithelial cells in vitro (Kuroki et al. 1991).

2.4.4.1 Interaction of SP-D with lipids

Even though SP-D does not interact with the major surfactant phospholipids, it binds in vitro to some other phospho- and glycolipids present in surfactant. SP-D interacts with the inositol and lipid moieties of PI and with glucosylceramide (Kuroki et al. 1992b, Ogasawara et al. 1992, Persson et al. 1992, Ogasawara et al. 1994, Sano et al. 1998, Saitoh et al. 2000). SP-D may compensate for SP-A deficiency and form surface-active tubular structures with SP-B if the appropriate PI ligand is present (Poulain et al. 1999) The interactions of SP-D with lipid ligands could contribute to surfactant lipid reorganization or the interactions of these molecules with host cells.

2.4.4.2 Interaction of SP-D with micro-organisms

SP-D has been shown to bind to a variety of bacteria, including rough strains of Salmonella Minnesota and E. coli as well as Klebsiella pneumoniae and Pseudomonas aeruginosa (Lim et al. 1994b). SP-D also stimulates the phagocytosis of Pseudomonas aeruginosa (Restrepo et al. 1999). The interaction of SP-D with bacteria often results in CRD-dependent bacterial aggregation, agglutination. Unlike SP-A (van Iwaarden et al. 1994), SP-D does not bind to lipid A. It interacts with E. coli through the core polysaccharides and/or the O-specific antigens. The core region of the LPS of other gram-negative bacteria is broadly recognized by SP-D as well (Kuan et al. 1992).

SP-D has been shown to bind to the influenza A virus, resulting in aggregation of the target (Hartshorn et al. 1996a). The binding and inhibition of hemagglutination was inhibited by chelation of calcium and by carbohydrates, suggesting that the interaction of SP-D with the virus was mediated via the CRD. SP-D also enhances the neutrophil uptake of the virus in a calcium-dependent manner (Hartshorn et al. 1997). Further

29

enhanced antiviral and opsonic activity for influenza A virus was obtained by making a human MBP and SP-D chimera (White et al. 2000). The degree of multimerization of SP-D also appears to be important for its interactions with viruses (Brown-Augsburger et al. 1996, Hartshorn et al. 1996b). SP-D induces massive aggregation of influenza A virus particles (Hartshorn et al. 1996a). This massive agglutination of organisms could contribute to lung host defence by promoting airway mucociliary clearance, but it could also promote internalization by phagocytic cells. Recombinant SP-D inhibited RSV infectivity both in vitro and in vivo (Hickling et al. 1999), and reduced SP-D protein levels have been detected in RSV infection (Kerr & Paton 1999).

A recent study documented direct interaction between the yeast Candida albicans and SP-D, thus further confirming the importance of SP-D in innate immunity (van Rozendaal et al. 2000).

2.4.5 SP-D receptors

SP-D binds with high affinity to alveolar macrophages. Miyamura and his co-workers (1994) reported SP-D binding to alveolar macrophages that was not calcium-dependent, whereas another study (Kuan et al. 1994) suggested that SP-D binds to alveolar macrophages in a saturable, concentration- and calcium-dependent manner. The binding could be inhibited by mannose.

An SP-D-binding protein, named gp-340, has been isolated from human alveolar proteinosis lavage (Holmskov et al. 1997). This protein is a member of the scavenger receptor family, it is calcium-dependent but not mannose-inhibitable, and it binds to the CRD of SP-D. This protein can also interact with SP-A (Tino & Wright 1999).

According to these studies, there is probably more than one specific binding site for SP-D in alveolar macrophages. Both SP-D and SP-A bind the CD14 LPS receptor (Sano et al. 1999). SP-D binds to the associated sugars, whereas SP-A binds to the protein backbone of CD14.

2.4.6 SP-D-deficient mice

Based on in vitro studies, which showed SP-D to have no direct effect on surfactant homeostasis, the SP-D-deficient mice were a surprise to the investigators. SP-D (-/-) mice have grossly normal lung function, but several significant perturbations of surfactant homeostasis were detected (Botas et al. 1998, Korfhagen et al. 1998). The deletion of SP-D gene expression alters the alveolar type II epithelial cell morphology, leading to hypertrophy of these cells and enlargement of lamellar bodies (Botas et al. 1998). Abnormalities in the morphology of alveolar macrophages, in pulmonary-associated lymphoid tissue and in lung structure were evident. The pool sizes of surfactant increased remarkably, leading to accumulation of lipids and alveolar macrophages in the alveolar space. Tubular myelin structures were not seen, but no effect on the surface activity of surfactant was detected (Korfhagen et al. 1998). The lipid accumulation could be

30

corrected with pulmonary-specific expression of SP-D (Fisher et al. 2000), suggesting a prominent role of SP-D in surfactant metabolism.

Thus far, there are only a few reports on the host defence functions of SP-D knockout mice. The clearance rates of Haemophilus influenzae were not perturbed in mice lacking SP-D. However, pulmonary inflammation was increased in SP-D-deficient mice compared with wild-type controls. The association of Haemophilus influenzae with macrophages decreased in the absence of SP-D, suggesting a defect in opsonization and/or phagocytosis (LeVine et al. 2000). Additionally, mice lacking SP-D were susceptible to both influenzae and RSV pneumonia. The clearance of the virus from the lung was impaired in SP-D-deficient mice (LeVine & Whitsett 2001).

2.4.7 SP-D in lung diseases

Similarly to SP-A, SP-D levels have been shown to be altered in many diseases, mainly infectious diseases. Allelic variants of SP-D have also been found (DiAngelo et al. 1999). Since SP-D was earlier thought to act mainly in host defence, no studies on SP-D levels contributing to ontogeny or lung function have been published. SP-D concentrations in amniotic fluid increase progressively with gestational age (Inoue et al. 1994).

Similarly to SP-A, increased BALF SP-D concentrations have been measured from patients with pulmonary alveolar proteinosis (Honda et al. 1995b), idiopathic pulmonary fibrosis (Honda et al. 1995b) and bacterial pneumonia (Limper et al. 1994). Also similarly to SP-A, SP-D concentrations detected from sera increase in these lung diseases (Honda et al. 1995b). Thus, the determination of serum levels of SP-A and SP-D may be useful in predicting the disease, even if the values do not correlate with physiological lung function tests.

In RSV infection, similarly to SP-A, the BALF SP-D concentration was reduced (Kerr & Paton 1999).

2.5 Surfactant Protein B (SP-B)

2.5.1 SP-B gene

SP-B is encoded by a single gene located on the short arm of human chromosome 2 (Emrie et al. 1988). The SP-B gene is approximately 9.5 kb long and composed of 11 exons, of which the 11th exon is untranslated. The SP-B gene is transcribed into a 2 kb mRNA. Mature SP-B is encoded by the exons 6 and 7 of the SP-B gene. SP-B cDNA sequences are highly conserved between species (Emrie et al. 1989, Pilot-Matias et al. 1989).

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2.5.2 SP-B expression and subcellular localization

In alveolar type II epithelial cells, SP-B precursors are detected in the endoplasmic reticulum, the Golgi complex and the multivesicular bodies, whereas mature SP-B is found in multivesicular and lamellar bodies, where it is stored and secreted into the alveolar space (Voorhout et al. 1992a). Apart from alveolar type II epithelial cells, SP-B is synthesized in nonciliated bronchiolar epithelial (Clara) cells (Phelps & Floros 1988). So far, SP-B has not been localized in Clara cells in vivo, and very little is known about the synthesis and processing of SP-B in these secretory cells. It is unclear if SP-B expression in Clara cells is related to surfactant function or some novel function. However, it seems that SP-B processing and function in Clara cells differ from those in alveolar type II epithelial cells (O'Reilly et al. 1989, Wikenheiser et al. 1992, Lin et al. 1999).

2.5.3 SP-B protein

Surfactant protein B is a hydrophobic peptide that avidly associates with surfactant phospholipids in the alveolar airspace. The primary translation product of human SP-B mRNA consists of 381 amino acids. Through a proprotein state of 42 kDa and further processing of a 23-kDa intermediate form, the mature hydrophobic peptide, containing 79 amino acids, is secreted into the alveolar lumen as a disulfide-linked dimer of 18 kDa (O'Reilly et al. 1989). SP-B processing is illustrated in Fig. 2.

Processing of the SP-B preproprotein to its mature peptide occurs during transit through the secretory pathway. The entry to this pathway is mediated by the N-terminal signal peptide, which is cleaved when the proprotein reaches the endoplasmic reticulum. N-terminal propeptide is needed for the transit of SP-B out of endoplasmic reticulum (Lin et al. 1996a), and both N-terminal propeptide and mature peptide are needed for the trafficking of SP-B further to lamellar bodies (Lin et al. 1996b). In contrast, it seems that the C-terminal peptide is not required for intracellular trafficking (Lin et al. 1996b).

The mature peptide shares sequence homology with the saposin proteins A-D, which establishes it as a member of the saposin-like protein family. Members of this protein family share a common secondary structure characterized by three conserved intramolecular cysteine bridges (Vaccaro et al. 1999). SP-B differs from the other members of this family in that it is more hydrophobic and forms sulfhydryl-dependent homodimers (Johansson et al. 1991). In humans and mice, the mature peptide is detected exclusively as a homodimer. However, the formation of SP-B dimers is not essential for surfactant function, although dimerization could optimize lung function (Beck et al. 2000a).

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Fig. 2. SP-B processing. The preproprotein consists of 381 amino acids. The amino-terminus is cleaved in the proprotein. In the further processing, the amino-terminal peptide and the carboxy-terminal peptide are cleaved to form the mature SP-B consisting of 79 amino acids. The Y symbol represents the glycosylation site of the protein. (Modified from O'Reilly et al. 1989).

2.5.4 Functions of SP-B related to diseases

Mature SP-B increases the surface adsorption and decreases the surface tension of surfactant phospholipids required for normal lung function (Beck et al. 2000b). Pulmonary surfactant insufficiency in newborn infants leads to RDS, a leading cause of morbidity and mortality among premature neonates worldwide. Substantial benefit is derived from treating affected infants with surfactant replacement preparations, particularly ones containing the hydrophobic surfactant proteins B and C. Hereditary SP-B deficiency in human infants results in fatal respiratory failure after birth (Nogee et al. 1993). Similarly, mice with deleted SP-B expression develop lethal respiratory failure at birth (Clark et al. 1995).

Studies on transgenic mice have further indicated the critical role of SP-B in the maintenance of pulmonary surfactant homeostasis. Lung morphogenesis and surfactant phospholipid synthesis in SP-B (-/-) mice proceed normally prior to birth (Clark et al. 1995). However, lamellar body formation is disrupted, resulting in abnormal inclusions

COOH

COOH

COOH

COOH

NH2

NH2

NH2

NH2

NH2 preproSP-B Mr=40 kDa

proSP-B Mr=39 kDa

amino-terminal peptide Mr=16 kDa

carboxy-terminal peptide Mr=23 kDa

mature SP-B Mr=8 kDa

1 23 200 279 381

COOH

33

consisting of multivesicular bodies and disorganized lamellae. Neither mature lamellar bodies nor tubular myelin were detected in SP-B (-/-) mice, and alveolar type II epithelial cells lost their polarity. However, the electron-dense masses in disrupted lamellar bodies were secreted into the airway (Stahlman et al. 2000). In addition to the effects on lamellar body biogenesis, SP-B deficiency resulted in aberrant processing of the SP-C proprotein, leading to accumulation of an 11-kDa processing intermediate and a decline of the mature SP-C peptide level (Clark et al. 1995).

Recently, it has been proposed that SP-B also contributes to the anti-inflammatory properties of surfactant (Miles et al. 1999). However, the available evidence is preliminary and the issue warrants further studies.

2.6 Surfactant Protein C (SP-C)

SP-C is the only SP that is pulmonary surfactant-specific and a constituent unique to pulmonary surfactant. No homologous proteins to SP-C have been identified thus far. SP-C is extremely hydrophobic and known to be expressed solely by alveolar type II epithelial cells. Clara cells of bronchiolar epithelium do not express SP-C (Kalina et al. 1992). The human SP-C gene is 3.5 kb in length and located in chromosome 8. The larger proprotein is processed to a 3.5-kDa mature peptide (Nogee 1998).

Although SP-C imparts important surface properties to surfactant phospholipid mixtures, the function of this protein in vivo is not fully understood. SP-C seems to associate with chronic lung disease, but no gross abnormalities were detected in SP-C knockout mice (Weaver & Conkright 2001).

2.7 Eustachian tube (ET)

The Eustachian tube (ET) is a passage which extends from the middle ear cavity behind the eardrum to the pharynx, measuring approximately 3 cm in human adults. A schematic representation of the location of the ET is shown in Fig. 3. The main function of the ET is to equalize middle ear pressure with atmospheric pressure. Usually, the ET is closed with its wall collapsed, which helps to prevent inadvertent contamination of the middle ear by the normal pharyngeal secretions. The ET opens during swallowing, yawning or chewing, allowing air to pass through. Similar to the remaining parts of the ET, its pharyngeal opening is collapsed at rest but opens briefly to allow exchange of gases between the middle ear and the nasopharynx, facilitating aeration of the middle ear space. (Monsell & Harley 1996.)

The ET develops embryonally from the first pharyngeal pouch and connects the upper respiratory tract to the middle ear. Apart from protecting the middle ear from excessive deviations of atmospheric pressure, the ET serves as a clearance tract and protects the middle ear against noxious agents from the airways. The ET is involved in all diseases concerning the middle ear. When active function of the tube is compromised, middle ear pressure becomes more negative in relation to atmospheric pressure. ET dysfunction may result in obstruction or in abnormal patency of the tube and thus contribute to the

34

development of middle ear infection. A persistent functional ET obstruction may eventually even result in atelectasis of the tympanic membrane. (Bluestone 1985.)

The ET prevents nasopharyngeal foreign material from entering the middle ear. The protective function is dependent upon the length of the tube, the radius of its lumen and the compliance of its walls. The local defence system in ET also plays an important role in preventing middle ear infections. Dysfunction of local mucosal immunity in the ET may predispose infants to recurrent otitis media, because the tube is the route for pathogens from the upper airways to the middle ear. (Bluestone & Klein 1995.)

The upper ET compartment is closely sheltered by cartilage, and the lower compartment has an elongated lumen with numerous inferior mucosal folds. The ventilatory function of the ET is accomplished by the pharyngeal muscles that pull apart the mucous surfaces of the cartilaginous ET (Mulder & Kuijpers 1991, Prades et al. 1998). The mucociliary blanket clears the secretions from the middle ear into the nasopharynx.

Fig. 3. Schematic representation of the human middle and inner ears. The ear is divided into three parts: the external ear, the middle ear and the internal ear. 1. The pharyngeal opening of the Eustachian tube. The middle ear consists of the tympanic cavity (8), the mastoid cells and the Eustachian tube (2). 3. The isthmus of the tube, where the bony and the cartilaginous parts of the Eustachian tube merge. 4. The bony part of the Eustachian tube heading towards the tympanic cavity. 5. The tympanic membrane. 6. The tensor tympani muscle. 7. The bony labyrinth of the internal ear. 9. The epitympanic recess. The external ear includes the auricle and the external acoustic meatus (10). (Adapted with permission from Kahle 1993).

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2.7.1 Cells in Eustachian tube epithelium

The mucosa of the fibrocartilaginous portion of the ET consists of ciliated pseudostratified epithelium of respiratory type, which rests on a basement membrane (Hiraide & Inouye 1983). The ET epithelium includes cuboidal epithelial cells with microvilli, defined as dark or intermediary granulated cells, columnar ciliated cells, goblet cells and basal cells (Fig. 4).

Ciliated and mucus cells are concentrated in the lower part of the cartilaginous ET, which contains numerous mucosal folds. In addition to ciliated cells, the mucociliary system contains secretory cells and a mucus blanket (Lim 1974). Ciliated cells are responsible for mucociliary transport; the ciliary function keeps the mucus in movement from the ET to the nasopharynx, preventing bacterial invasion into the middle ear (Hussl & Lim 1969).

Non-ciliated granulated cells are considered secretory (Lim & Shimada 1971). They differ in size, shape and the morphological characteristic of their secretory granules (Lim 1974). Intermediary and dark granulated cells differ in shape: some of them resemble goblet cells, while others are columnar or cuboidal. The secretory granules can be detected apically and the nucleus basally. The surface of intermediary cells and dark granulated cells often bulges towards the lumen (Hussl & Lim 1969). Similar electron-dense granules have been demonstrated in mucous cells of the gastric mucosa as well as in secretory cells of the human tracheal epithelium (Hussl & Lim 1969). Yet today, the secretory products of these cells are poorly understood. It has been proposed that goblet, intermediary and dark granulated cells may be different phenotypes of the same secretory cell (Hussl & Lim 1969).

Basal cells are generally considered undifferentiated cells, which become one of the epithelial cell types as they undergo differentiation (Lim 1974).

All mucosal surfaces, including ET, secrete mucus, which contains mainly mucins, high-molecular weight glycoproteins. Mucins protect and lubricate the epithelial surface and trap bacteria and viruses for mucociliary clearance (Lim et al. 1991). It has also been proposed that serous ET epithelial cells together with serous glands secrete antimicrobial peptides, which contribute to the innate immunity system of the ET. The biology of ET epithelial cells and the products secreted by these cells could play a critical role in the protection of the ET and the middle ear against microbial invasion (Lim et al. 2000). However, these issues are not well known thus far.

36

Fig. 4. Cells in the middle ear mucosa. The epithelium is similar in the Eustachian tube. The ET epithelium consists of columnar ciliated cells, cuboidal secretory epithelial cells with microvilli, defined as dark or intermediary granulated cells, goblet cells and basal cells. (Reproduced with permission from Lim 1974).

2.7.2 Eustachian tube and otitis media

Otitis media is an inflammation of the middle ear. Subcategories include acute otitis media, otitis media with effusion and chronic otitis media. Otitis media with effusion is also called secretory otitis media or glue ear. Infections in the middle ear are among the most common illnesses during childhood. They affect nearly all children at least once, and about 20 % of children suffer from recurrent otitis media (ROM). In Finland, by 2 years of age, over 50 % of children have had middle ear infection at least once, and by 5 years of age, over 80 % of children have had otitis media. Thus, middle ear infections are a remarkable socio-economic problem. (Karma 1999.)

The pathogenesis of otitis media is complicated and multifactorial. The infection starts from the upper airways and spreads through the ET, leading to otitis media. The closure of the ET results in negative pressure in the middle ear. The presumed dysfunction of

37

ciliary activity further facilitates the accumulation of effusion in the cavity. Under these circumstances, extension of the upper airway infection into the middle ear is likely. Normal tubotympanal mucosa is protected by non-specific defensive agents, such as mucus, lactoferrin and lysozyme, and additionally by specific immune systems, such as surface mucosal immunoglobulin, which inhibit bacterial adhesion to the mucosal surfaces (Lim et al. 1987).

ET dysfunction is considered a principal pathogenic factor in susceptibility to ROM, which is presumed to be largely determined by hereditary factors (Bluestone 1996). With clinical tests measuring pressure equilibration in the middle ear, the site of dysfunction has been localized in the distal part of the cartilaginous portion of the tube (Takahashi et al. 1987). Additionally, pre-existing good ET function (measured by tympanometry and sonotubometry) has been found to reduce the otological complications of viral upper respiratory tract infections (Doyle et al. 2000). Active ET function seems to be weaker in children with otitis media with effusion compared to healthy children. Furthermore, active ET function in common is impaired in children compared to adults (Bylander-Groth & Stenstrom 1998). The ET of small children is shorter compared to older children and adults, and the cartilage supporting their ET is stiff and less prominent. Moreover, the tensor veli palatini muscle, which opens the tube, is weaker in infants. These structural differences that lead to impaired function of the tube could explain the proneness of infants to middle ear infections (Bluestone 1985). However, the actual mechanisms and potential genes involved in recurrent middle ear infections are unknown.

2.7.3 Pathogens involved in middle ear infections

Otitis media is generally considered a simple bacterial infection that can be effectively treated with antibiotics. However, only in about 70 % of the cases of acute otitis media can pathogenic bacteria be isolated from middle ear fluid. Thus, it has been shown that respiratory viruses also play a crucial role in the aetiology and pathogenesis of acute otitis media. Infections may also be combined viral and bacterial infections. (Heikkinen et al. 1999.)

An infection of the upper respiratory tract initiates a whole cascade of events, which eventually lead to the development of acute otitis media as a complication. Respiratory viruses induce a release of inflammatory mediators in the nasopharynx. Viral inflammation may enhance the invasion of bacteria from the nasopharynx to the middle ear by provoking sniffing, sneezing and ciliary and ET dysfunction. This perturbation of the cleaning functions of the ET mucosa leads to the suppression of the host’s immune defence (Heikkinen & Chonmaitree 2000).

The predominant pathogens of recurrent and persistent acute otitis media are antibiotic-resistant Streptococcus pneumoniae (in 30–50% of cases) and betalactamase-producing Haemophilus influenzae (in 10-20% of cases) (Bluestone et al. 1992). These same pathogens also cause respiratory infections, sinusitis and pneumonia. The respiratory viruses that primarily play a crucial role in the pathogenesis of otitis media are RSV and influenza viruses (Chonmaitree & Heikkinen 1997). Different types of respiratory viruses may vary in their ability to predispose to otitis media. Also, some

38

viruses may enter the middle ear passively along with nasal secretions, whereas other viruses actively invade the middle ear and contribute to the inflammatory process in the middle ear mucosa (Heikkinen et al. 1999).

The viral infections of the upper respiratory tract may have a substantial impact on the bacterial colonization of the nasopharynx and the adherence of bacteria to epithelial cells. For example, RSV infection significantly enhances the attachment of Haemophilus influenzae to human respiratory epithelial cells (Jiang et al. 1999).

2.7.4 Surfactant in Eustachian tube

It is believed that surface-active agents facilitate the opening of the ET. Several studies have tentatively shown that ET lavage fluid (ETLF) contains surface-active material (Birken & Brookler 1972, Lim 1974, Hills 1984, Wheeler et al. 1984, Coticchia et al. 1991). Phospholipid-containing lamellar structures within the epithelial cells and extracellular spaces of rabbit ET have been detected (Mira et al. 1988), and Karchev and his co-workers (Karchev et al. 1994) reported similar findings in mouse ET. The proposed function for surface-active agents in ET has been to lower the surface tension, thereby facilitating normal opening of the tube. Indeed, surfactant has been described to reduce the pressure required to force the ET open in ear-infected rats (White et al. 1990). A similar effect of intranasally aerosolized synthetic surfactant was recently detected in a gerbil model (Venkatayan et al. 2000). The duration of infection was also decreased by the surfactant treatment. Additionally, administration of Ambroxol, which enhances surfactant production, to patients with secretory otitis media has resulted in a decreased prevalence of otitis media with effusion (Passali & Zavattini 1987).

SP-A has been detected with immunoassay in middle ear effusions of patients with otitis media. The study described SP-A presence in serous effusions in most cases, but less often in purulent, mucoid or hyperviscous effusion fluids, respectively (Yamanaka et al. 1991). In 1992, Kobayashi and his co-workers reported of a water-soluble 80-kDa protein in the human middle ear, which cross-reacted with monoclonal antibodies to human SP-A (Kobayashi et al. 1992). Recently, SP-A expression has also been detected in rabbit middle ear mucosa (Dutton et al. 1999).

3 Outlines of the present study

The putative surfactant system in the ET has been a topic of discussion for over 25 years. The present study is based on a study from the early 90’s, which showed SP-A immunoreactivity in human middle ear epithelium. The increasing evidence of SP-A and SP-D involvement in the pulmonary host defence system and in interactions with bacteria causing middle ear infections raised a question about the expression of surfactant proteins in ET. The local defence system could be critical in preventing ear infections.

Both ET and lung epithelia are of endodermal origin and the ET epithelium resembles that of the lower airways. The opening and closure of the ET are important for tube function, and it is likely that some kind of anti-adherent material facilitates this function.

In order to increase our basic knowledge of the putative ET surfactant system, the following goals were set for this work:

1. To determine whether surfactant proteins are expressed in ET. During this study it was discovered that SP-A, SP-B and SP-D are expressed in

porcine ET, and further goals were therefore set: 2. To localize the mRNA expression and surfactant proteins within ET epithelium. 3. To characterize the phospholipids and surface activity of ET lavage fluid (ETLF)

and to compare them with those of bronchoalveolar lavage fluid (BALF).

4 Materials and methods

Detailed descriptions of the materials and methods have been given in the original articles I-IV.

4.1 Eustachian tube preparations and bronchoalveolar lavage

4.1.1 Preparation of total RNA (I, II)

The pharyngeal openings of porcine and ovine ETs were prepared, and a 20 G catheter was inserted into the tube. RNA was recovered from the ET lining by lavaging the tube with phenol-based RNA-STAT 60 (Tel-Test, Inc.). Total RNA was isolated from the lavage return and subsequently used for RT-PCR and Northern blot analysis. Total RNA from porcine lung was isolated as control. Later, total RNA was isolated directly from ET epithelial cells. The ET was dissected and opened and the epithelial cells were scraped with a scalpel.

4.1.2 Preparation of samples for light microscopic and protein studies

(II, III)

The pharyngeal opening of the ET was prepared, and approximately 1 cm of the cartilaginous ET was dissected. The samples were fixed overnight and embedded in paraffin. 4 µm cross-sections were mounted on SuperFrost Plus (Menzel-Gläser, Braunschweig, Germany) slides and used for immunohistochemistry and in situ hybridization studies. For protein analyses, ETs were lavaged with a catheter and a syringe, as described, with 1 x PBS. Bronchoalveolar lavage (BAL) was performed as control. Briefly, the trachea was cannulated and the airways were filled with 0.9% saline

41

at a pressure of 30 cm H2O. The fluid was collected from the airways using gentle suction. The procedure was repeated three times and the lavage returns were combined.

4.1.2.1 Preparation of aggregate fractions (II, IV)

The ETs of approximately 200 pigs from a slaughterhouse were lavaged as described. As control, porcine lungs were lavaged as described above. After separation of cells with centrifugation at 500 x g for 10 minutes, the samples were re-centrifuged for two hours at 100 000 x g to collect the sedimentable lipid-protein complexes. The pellets obtained were brought to suspension and fractionated on a discontinuous sucrose density gradient. The samples were centrifuged and the aggregate layer of BALF and the comparable layer of ETLF were collected and diluted with 0.15 M NaCl. The centrifugation was repeated, and the pellets were collected.

For Western analyses of SP-A and SP-D, the ET and bronchoalveolar lavage returns were centrifuged and the sedimentable lipid-protein complexes and the supernatant fractions were collected and analyzed.

4.2 SP expression (I, II, III)

4.2.1 Cloning of cDNA fragments for ET SP-A, SP-D and SP-B (I, II)

Altogether 2 µg of total RNA was used for the RT-PCR reactions for SP-A, SP-D and SP-B. For SP-A and SP-B, the primers used were porcine-specific. For SP-D, degenerative primers were used. The RT-PCR reactions were performed using the Masteramp RT-PCR kit (Epicentre, Madison, WI) with 1 x enhancer in a one-tube reaction. The resulting fragments were purified and ligated into the pGEM-T Easy vector (Promega, Madison, WI) or the pBluescript KS (Stratagene) vector. The clones obtained were identified by sequencing on both strands.

4.2.2 Northern analysis (I, II)

The probes used for the expression studies are shown summarized in Table 2. For SP-A and SP-D, altogether 2 µg of lung total RNA and 10 µg of ET total RNA were run on a formaldehyde gel. For SP-B, 0.5 µg of lung RNA and 30 µg of ET RNA were used. The samples were transferred overnight onto a Biodyne B membrane (Pall Gelman Laboratory, MI). For Northern hybridization, SP-A and SP-D antisense 32P-dCTP-labelled riboprobes were synthesized using T7 RNA polymerase (Promega) or Sp6 RNA polymerase (Promega). The SP-B cDNA fragment from porcine lung was labelled directly with 32P-dCTP. The labelled products were purified on a Sephadex G-50 column

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(Amersham Pharmacia Biotech). The hybridizations were performed at 42°C overnight followed by high-stringency washes.

Table 2. The probes for the expression studies (I-III).

Probe Source Length (bp) Vector Usage PSP-A Porcine lung 365 (CRD) pBS-SK (EcoRV)

(Stratagene)

Northern analysis,

ISH

PSP-D Porcine lung 313 (CRD) pGEM-T Easy

(Promega)

Northern analysis,

ISH

OSP-D Ovine ET 304 (CRD) pGEM-T Easy

(Promega)

PSP-B Porcine lung 231 pGEM-T Easy

(Promega)

Northern analysis,

ISH

4.2.3 In situ hybridization (II, III)

In situ hybridization (ISH) was carried out basically according to the instructions supplied by Boehringer-Mannheim (Mannheim, Germany). Briefly, the 365 bp SP-A, 313 bp SP-D and 231 bp SP-B cDNA fragments of porcine lung were subcloned into the pBluescript (Stratagene, La Jolla, CA, USA) and pGEM-T Easy (Promega, Madison, WI, USA) vectors. The plasmids were linearized, and the antisense and sense UTP-digoxigenin (DIG) -labelled riboprobes were synthesized using T7 RNA polymerase (Promega), T3 RNA polymerase (Promega) or Sp6 RNA polymerase (Promega). After DNase I digestion, the probes were purified with lithium chloride-ethanol precipitation and detected on agarose gel. The labelling was detected with the DIG detection kit supplied by Boehringer-Mannheim.

Four-µm paraffin sections were treated with proteinase K and postfixed. The sections were acetylated, washed and covered with prehybridization mix followed by hybridization mixture containing 300 ng/ml of the digoxigenin-labelled riboprobe. Hybridization was allowed to occur at 58ºC for 42 hours. After hybridization, the unbound probe was removed from the sections by treatment with RNase A followed by high-stringency washes. The hybridized probe was detected by incubating the sections with the antidigoxigenin antibody conjugated with alkaline phosphatase. The colour reaction took place overnight with nitroblue-tetrazolium 5-bromo-4-chloro-3-indolyl phosphate (Boehringer-Mannheim). The sections were counterstained with 0.02% FCF fast green (Sigma). As a positive control, sections from porcine lung were hybridized with the same riboprobes. Negative controls consisted of the same tissue sections hybridized in an identical fashion with the digoxigenin-labelled sense riboprobe. As another negative control, tissue sections hybridized with the antisense riboprobe were subjected to a colour reaction without anti-digoxigenin antibody incubation.

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4.3 Microscopy studies (I, II, III)

The antibodies and their usage in the protein studies are shown summarized in Table 3.

Table 3. The antibodies for the protein studies (I-III).

Antibody

Definition Source, reference Usage

PE-10 Monoclonal mouse anti-

human SP-A

Prof. T. Akino,

Sapporo, Japan

Immunohistochemistry

Western analysis

IEM

SheepSP-A Polyclonal rabbit anti-

ovine SP-A

Prof. S. Hawgood, San

Francisco, USA

Immunohistochemistry

Western analysis

IEM

PigSP-D Polyclonal rabbit anti-

porcine SP-D

Dr. M. van Eijk,

Utrecht, The

Netherlands

Immunohistochemistry

Western analysis

IEM

RatSP-D Polyclonal rabbit anti-

rat SP-D

Prof. E. Crouch, St.

Louis, USA

Western analysis

PigSP-B Monoclonal mouse anti-

porcine SP-B

Dr. Y. Suzuki, Kyoto,

Japan (Suzuki et al.

1986)

Immunohistochemistry

Western analysis

IEM

SheepSP-B Polyclonal rabbit anti-

ovine SP-B

Prof. S. Hawgood, San

Francisco, USA

Immunohistochemistry

Western analysis

IEM

4.3.1 Immunohistochemistry (I, II, III)

The isolated ETs were fixed in 4% paraformaldehyde and embedded in paraffin. The cross-sections were deparaffinized and rehydrated. Antigen retrieval was performed in trypsin or by heating in citrate-phosphate buffer. Endogenous peroxidase was blocked with aqueous H2O2. An avidin-biotin-peroxidase method was applied. The sections were incubated with primary antibodies. For SP-A, monoclonal human anti-SP-A antibody PE-10 or polyclonal sheep anti-SP-A antibody was used. For SP-D, polyclonal porcine anti-SP-D antibody was used, and for SP-B, monoclonal porcine anti-SP-B or polyclonal sheep anti-SP-B antibody was used. After the addition of the secondary antibody, the sections were treated with the avidin-biotin-peroxidase complex (Dakopatts a/s, Glostrup, Denmark DAKO). Diaminobenzidine (Sigma, St. Louis, MO, USA) or AEC complex was used as the chromogen, and the sections were slightly counterstained with Mayer’s hematoxylin. PBS replaced the primary antibody as a negative control. Sections of porcine lung tissue served as positive controls.

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4.3.2 Electron microscopy (II, III)

For electron microscopy, tissue pieces from porcine ET (approximately 1 mm in diameter) were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. They were then post-fixed in 1% osmium tetroxide in phosphate buffer, dehydrated in acetone and embedded in Epon LX 112. Thin sections were cut with a Reichert Ultracut E microtome and examined under a Philips CM 100 transmission electron microscope using an acceleration voltage of 80 kV.

4.3.3 Immunoelectron microscopy (II, III)

Fresh porcine ET and lung tissue were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer. Small tissue pieces were immersed in sucrose and frozen in liquid nitrogen. Thin cryosections were cut with a Leica Ultracut UCT microtome. For immunolabelling, the sections were first incubated in 5% BSA in PBS. Then the sections were incubated with the primary antibodies. After washings, the sections were exposed to secondary antibodies, followed by a protein A - gold complex. The controls were prepared by carrying out the labelling procedure without the primary antibody. The sections were embedded in methylcellulose and examined as described above.

4.4 Western analysis (II, III)

SP-D was analyzed from the supernatant fraction and SP-A from the sedimentable lipid-protein aggregate fraction of ETLF and BALF. The supernatants obtained were concentrated, and the protein concentrations were determined. The samples for SP-A and SP-D were reduced according to Laemmli (1970) and separated by 12% SDS-PAGE gel. For SP-B analysis, the unreduced samples were separated by 15% SDS-PAGE gel. Electrophoresed samples were blotted onto a nitrocellulose membrane, blocked and incubated with primary antibodies. The secondary antibodies were visualized by chemiluminescence (ECL Plus Western Blotting Analysis System, Amersham, Buckinghamshire, UK).

4.5 Analysis of phospholipid molecular species by electrospray

ionization mass spectrometry (IV)

Molecular species compositions of phospholipid were analysed by ESI-MS using a Micromass Quatro Ultima triple quadrupole mass spectrometer (Micromass, Wythenshaw, UK) equipped with an electrospray ionization interface. Total lipid was extracted from ETLF and BALF according to Bligh and Dyer (1959). Prior to extraction, internal recovery standards were added. The samples were dissolved in

45

methanol:chloroform:water and introduced into the mass spectrometer by nanoflow infusion. The PC species were detected by positive ionization, while PI and other acidic phospholipids were preferentially detected using negative ionization. The data were acquired and processed using the MassLynx NT software. After conversion into centroid format according to area and correction for 13C isotope effects and for the reduced response with increasing m/z values, the PC and PI species were expressed as percentages of their respective totals present in the sample. The predominant molecular species present for each ion peak resolved was determined by analysis of the fatty acyl fragments generated by collision gas-induced fragmentation under negative ionization.

4.6 Surface activity measurements (IV)

The surface tensions of ETLF and BALF preparations were measured using a pulsating bubble surfactometer (Electronetics Co., Amhurst, NY). The concentrations of BALF and ETLF were adjusted on the basis of the PC concentration. The radius of the bubble was varied between 0.4 and 0.55 mm at a frequency of 0.33 Hz for 5 min at 37oC for 3 minutes. The surface tensions during the pulsation were recorded. For the estimation of surface adsorption and the equilibrium surface tension measurements, a bubble with a radius of 0.4 mm was formed and the radius was maintained constant for 5 min. The surface tensions were continuously measured during the first 10 seconds and thereafter at set time points. Surface tension at 5 min was called the equilibrium surface tension.

5 Results

5.1 Expression of SP-A, SP-B and SP-D in ET (I, II, III)

The resulting 365 bp SP-A cDNA fragment of porcine ET revealed 100% homology compared to the fragment obtained from porcine lung. SP-A mRNA of approximately 2.0 kb was detected in porcine lung and 1.8 kb in ET.

A 313 bp SP-D cDNA RT-PCR product was obtained from porcine lung and ET. Ovine lung and ET RNA showed a 304 bp fragment. The fragments were highly conserved. The sequences were identical to those detected in lung. The porcine sequence showed an insertion of three amino acids compared to the other species. The SP-D mRNA was approximately 1.4 kb in both ET and lung.

The 231 bp SP-B cDNA fragment obtained from ET revealed 100% homology compared to the fragment obtained from porcine lung. SP-B mRNAs of two different sizes could be detected in porcine ET. The other messenger was of the same size, i.e. approximately 2.5 kb, and it was detected from the RNA preparation isolated from porcine lung tissue. The other band detected was smaller, approximately 1.2 kb.

To identify the cellular sites of SP mRNA expression in porcine ET, a series of in situ hybridizations with porcine ET sections was performed. The expressions were studied throughout the circumference of the epithelium in the middle segment of ET. The pharyngeal and more distal parts of the ET were similarly studied. Using specific RNA probes for SP-A and SP-D, intense labelling with the antisense probes was seen in some epithelial cells near the mucosal folds in the floor of the tube lumen. The labelling of SP-A was more intense and generalized among the ET cells than the labelling with the SP-D probe. SP-A mRNA was detected basally and intermedially, while only light staining could be seen apically. Similarly to SP-A, strong SP-D labelling was detected basally and in the intermediate part of the epithelium. Unlike SP-A, SP-D mRNA was additionally detected in the apical cells. The expression patterns were similar in the pharyngeal and more distal parts of the ET.

SP-B expression was detected in different parts of the ET, but mostly close to the mucosal folds in the lower compartment of the ET lumen. The expression was

47

concentrated in the cells between the mucosal folds and the narrow ET lumen within the epithelial cell lining of the ET.

No SP-C expression in ET was detected with RT-PCR or Northern analysis.

5.2 Localization of SPs in ET (I, II, III)

The porcine ET is a curved tube. The dorsal part is closely sheltered by cartilage, and the lower segment has an elongated lumen with numerous inferior mucosal folds. The ET epithelium consists of stratified epithelium that morphologically resembles the lower airway epithelium. The ET epithelium could be described as columnar ciliated epithelium with some cuboidal epithelial cells. Goblet cells, ciliated cells and cells with microvilli are present. The apical areas of cells with microvilli contained electron-dense secretory granules.

The immunostainings were studied throughout the circumference of the epithelium in the middle segment of the ET. The pharyngeal and more distal parts of the ET were similarly studied. Light microscopic studies showed both SP-A and SP-D staining concentrated close to the mucosal folds. Immunopositivity of ET epithelial cells with both the anti-human and the anti-sheep SP-A antibody was detected. With the anti-sheep SP-A antibody, the labelling was localized to specific epithelial cells. Strong SP-A staining was detected apically in secretory cells. Goblet cells also showed immunopositivity for SP-A, as did some cells in the intermediate part of the epithelium. The SP-D antibody revealed intense reactivity in the epithelial cell lining, showing diffuse staining throughout the ET epithelium. However, some cells showed more intense staining both apically and basally. The apical staining for SP-D was stronger and more diffuse than that for SP-A, for which the positivity was specifically located in apical cells. The intermediate cells in the epithelium showed immunopositivity for both SP-A and SP-D. Faint diffuse SP-B staining was seen throughout the ET epithelium. With SP-B, in some cells, however, the apical parts showed a stronger, granular staining pattern. The immunostaining patterns were similar throughout the ET from the pharyngeal orifice to the more distal parts.

Immunoelectron microscopy confirmed the light microscopic findings. SP-A was localized in the microvillar epithelial cells, more specifically in the electron-dense material of apically located granules. Furthermore, in the granules of goblet-like cells, lamellar structures were detected and this material showed positive labelling for SP-A. More diffuse labelling was detected in the dark long microvillar cells. Traces of the SP-A label could also be seen in the ET lumen attached to the lipid material or to the microvilli of apical cells. The few macrophages found within the epithelial layer also contained SP-A.

The most prominent labelling with SP-D was seen in the apical plasma membrane and in the amorphous material lying on the epithelial cells. Few epithelial cells with microvilli showed diffuse labelling with the SP-D antibody. In these cells, secretory granules had no detectable immunoreactivity.

Electron microscopic studies of ET epithelium revealed SP-B label in small granules in cells with short apical microvilli. SP-B was also seen in association with electron-

48

dense material at the periphery of large apical granules. In these cells, the label was similarly seen to be attached to microvilli. Some lamellar structures were occasionally seen in extracellular spaces and rarely in epithelial cells.

A comparison of the SP-A, SP-D and SP-B expression and protein localization patterns is presented in Table 4.

Table 4. Comparison of SP-A, SP-D and SP-B expression and localization within ET epithelium.

Method* Localization SP-A SP-D SP-B In the circumference

of epithelium

Tubal floor and

mucosal folds

Tubal floor and

mucosal folds

Near mucosal folds ISH

Within the epithelial

cell layer

Mainly basal Basal and apical Basal and

intermediate

In the circumference

of epithelium

Tubal floor Diffuse Diffuse IHC

In specific cells Apically in

intermediary and

goblet cells

In some cells both

apically and basally

Apical granular

staining in

intermediary cells

Intracellular Apical secretory

granules of

microvillar

intermediary and

goblet cells

Endoplasmic

reticulum of specific

dark microvillar cells

Apical secretory

granules of

microvillar

intermediary cells

IEM

Extracellular Attached to

microvilli or lipid-

like material

Attached to secreted

amorphous material

in ET lumen

Attached to

secreted material

and traces

intercellularly

*The methods used are as follows: ISH = in situ hybridization, IHC = immunohistochemistry, IEM =

immunoelectron microscopy

5.3 Immunoreactivities of ETLF (II, III)

We found the characteristic monomeric SP-A protein triplet of 30-38 kDa in ETLF. The immunoreactivity of ETLF was similar to that detected in BALF. The antibody directed against sheep pulmonary SP-A also labelled the 28-kDa protein in the lipid-aggregate fraction of ETLF and BALF.

The antibody against porcine SP-D revealed intense labelling of the 48-kDa protein in the non-sedimentable fractions of ETLF and BALF.

49

Immunoreactivity with the SP-B antibody was detected both in ETLF and in the aggregate fraction isolated from ETLF with sucrose density gradient centrifugation. The size of the protein recognized by the porcine SP-B antibody was approximately 24 kDa, which is characteristic of the unreduced SP-B dimer. A similar major immunoreactive protein was detected in the specimens from BALF. The polyclonal antibody against sheep SP-B also showed positive reactions for the reduced samples, suggesting the presence of the mature 8-kDa form and the dimeric 18-kDa form of SP-B.

5.4 Phospholipid composition of ETLF (IV)

The distribution of phospholipid classes was very different in ETLF compared to BALF. PC comprised 92% of total phospholipids in BALF but only 66% of ETLF phospholipids, while PE was considerably enriched in ETLF (33% total phospholipids) compared to BALF (2% total phospholipids). Importantly, PG was present as the second major component in BALF phospholipids (6%), but was undetectable in ETLF. All phospholipid classes and the molecular species compositions of all fractions of varying density from either BALF or ETLF were essentially identical to those seen in the total lavage fluid. Moreover, the phospholipid composition of the underlying ET epithelial cells was very similar to that of the ETL fluid.

The molecular species compositions of all the phospholipid classes of ETLF were very different from those of BALF. The dominant PC species from BALF was PC16:0/16:0, which is the characteristic species of lung surfactant, while the remainder was a combination of saturated species. By contrast, PC16:0/16:0 was a minor component of ETLF, which was composed primarily of monounsaturated and diunsaturated species. While PC16:0/18:1 was common to both spectra, ETLF PC contained many species not present in BALF PC: ETLF contained significant amounts of species with sn-1 stearate (PC18:0/18:1) and sn-1 oleate (PC18:1/18:1) in contrast to the BALF PC species, which were all sn-1 palmitate. Importantly, arachidonoyl-containing species contributed appreciably to ETLF PC but not to BALF PC (PC16:0/20:4 and PC18:0/20:4). Finally, ETLF but not BALF contained a measurable contribution from sphingomyelin (SM16:0).

In common with the PC compositions, the spectrum of the PE species present in ETLF was completely different from that of BALF PE. BALF PE was composed almost exclusively of unsaturated diacyl species. In contrast, the PE species from ETLF were largely ether-linked rather than acyl-linked.

As expected, PG species were evident in BALF and had a monounsaturated and disaturated composition comparable to that described for other animal species. However, no corresponding ion peaks were detected in ETLF. For BALF, PI was essentially completely monounsaturated, while PS species were barely detectable. In contrast, the predominant acidic phospholipid species in ETLF were PS18:0/18:1 and polyunsaturated PI (PI18:0/20:4).

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5.5 Surface activity of ETLF (IV)

As expected, BALF revealed typical characteristics of lung surfactant. These included a very low surface tension approaching 0 mN/m during compression of the surface, marked hysteresis (surface tensions were much lower during compression than during expansion), rapid surface adsorption from the subphase and a low equilibrium surface tension around 23-25 mN/m. In sharp contrast to lung surfactant, the material from ET was not surface-active. ETLF had a minimum surface tension barely below 40 mN/m and low hysteresis during the pulsation of the bubble. There was an instantaneous lowering of surface tension after the formation of the bubble, with an equilibrium surface tension around 44 mN/m.

6 Discussion

This study shows, for the first time, that SP-A, SP-B and SP-D are expressed in ET. The expression and proteins are localized within the epithelium, and the phospholipids and the properties of ET surfactant are compared to those of alveolar surfactant.

The animal used in this study was pig. The ET of pig is big enough and resembles closely that of human. Pig has been described as a potential model for human middle ear infections. Otitis media is, to a large extent, dependent on the anatomical and physiological similarities between the ETs and middle ear cavities of pig and man. Middle ear inflammation has been described in pigs but appears to be infrequent (Pracy et al. 1998). The animals used in the present study were free of respiratory infections, and no signs of ear infections were recognized. In histological studies, no inflammatory changes were evident in the ETs studied.

At first, a technique to recover ET lavage fluid, which is similar to bronchoalveolar lavage fluid, was developed. The pharyngeal opening of the ET was prepared and the lavaging was performed with a cannula and a syringe. For phospholipid analyses, the ETLs from approximately 300 pigs were lavaged in one lot, the total number of lavaged animals approaching 1000.

The main problem was the availability of species-specific cDNA sequences and appropriate antibodies. The only porcine SP cDNA sequence found in the database was that of SP-A, while the other SP cDNA sequences were determined using degenerative primers. Also, a number of antibodies were tested to find the ones suitable for the studies.

With RT-PCR, Northern analyses and in situ hybridizations, expressions of SP-A, SP-D and SP-B were detected in the porcine ET epithelium. The cDNA sequences obtained from ET showed 100% homology compared to the sequences obtained from porcine lung. The porcine-specific probes obtained with RT-PCR were used for Northern analyses and in situ hybridizations. No expression for SP-C was detected in ET. Either the amounts of SP-C in ET are undetectable or SP-C is not expressed by ET epithelial cells. This would not be surprising, as SP-C has not been detected in Clara cells, either. No antibodies for SP-C were available because of its marked hydrophobicity, and immunostainings were therefore not studied.

The expression of SP-A was prominent near the mucosal folds of the epithelium. Previously, in addition to alveolar type II epithelial cells and Clara cells (Voorhout et al.

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1992b), SP-A expression has been detected in the epithelium of the conducting airways and in the tracheal and bronchial glands of the lower airways (Endo & Oka 1991, Khoor et al. 1993, Wong et al. 1996). SP-A mRNA has also been shown to be expressed in rat intestinal epithelium (Rubio et al. 1995) and human colon (Eliakim et al. 1997) as well as in prostate and thymus (Lu 1997) and, recently, also in middle ear epithelium and paranasal sinuses (Dutton et al. 1999). The function of SP-A in these tissues is not known, but it has been suggested to play a role in local antimicrobial host defence systems. According to the current knowledge, the role of SP-A in lung host defence is more significant than previously thought (Crouch & Wright 2001).

Light microscopy localized the SP-A protein close to the mucosal folds. The immunopositivities of ET epithelial cells with anti-human and anti-sheep SP-A antibodies showed slight differences. With the anti-sheep SP-A antibody, the labelling was localized to specific epithelial cells. Marked SP-A staining was detected apically in secretory cells. Additionally, goblet-like cells showed immunopositivity for SP-A.

Immunoelectron microscopy localized SP-A in the electron-dense granules of microvillar epithelial cells. In goblet-like cells, some of the lamellar structures detected also showed positive labelling for SP-A. In Clara cells in rat lung, SP-A has been detected in immature and mature secretory organelles (Voorhout et al. 1992b). The secretory organelles detected in ET epithelial cells are likely to secrete their lipid-like contents, as well as SP-A, into the ET lumen.

The expressions of both SP-D and SP-A were located near the mucosal folds in the ET epithelium. SP-D expression was detected throughout the epithelium, ranging from some basal to some apical cells. Besides alveolar type II epithelial cells and Clara cells, SP-D mRNA is also detected in the epithelium of the conducting airways and in both serous and mucous cells of the tracheal glands (Wong et al. 1996). Recently, SP-D expression was found in several human mucosal tissues, suggesting a prominent and more widespread role for SP-D within the mucosal surfaces. The SP-D protein was evident in the ET lumen, on the surface of apical cells, and attached to the plasma membrane. Similar labelling of amorphous airspace material in the lung has also been previously reported (Crouch et al. 1991). The pattern of immuno-gold labelling suggests that cellular SP-D is associated with secreted material rather than organelles involved in storage or degradation.

SP-B mRNA expression concentrated in the epithelial cells close to the mucosal folds in the narrow part of the ET lumen. Northern analyses, RT-PCRs and in situ hybridizations suggest that the expression of SP-D in ET epithelial cells was more abundant than the expression of SP-A or SP-B. The SP-B expression level detected was very low. These results may also be due to the different penetrations of probes into the cells or to differences in the binding of PCR primers. However, this pattern of SP abundance in ET was evident in protein studies as well.

In light-microscopic studies, faint SP-B protein staining was detected throughout the epithelium. However, more specific apical staining was confirmed by immunoelectron microscopy. The SP-B immunolabel was located in the apical granules and attached to the microvilli of cuboidal epithelial cells. SP-A co-localized with SP-B in the microvillar cells of apical secretory granules. However, the granules of goblet-like cells were free of SP-B labelling. These granules had some similarities in size with the lamellar bodies of alveolar type II epithelial cells. Other research groups (Lim 1974, Mira et al. 1988,

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Karchev et al. 1994) have reported the presence of lamellar bodies, similar to those seen in alveolar type II epithelial cells, in ET epithelial cells of guinea pig, mouse and rabbit. No such morphology was detected in porcine ET epithelial cells in immunoelectron microscopy. Although no typical lamellar structures were seen in the cryosections, a few concentric lamellar structures were detected in the apical cytoplasm of epithelial cells in conventional Epon sections. The lack of lamellar structures may be due to a cryotechnique, which is not considered equally good for preserving lipid structures as freeze substitution (Voorhout et al. 1991b). However, both cryotechniques and freeze substitution were used, and lamellar bodies were found in alveolar type II epithelial cells but not in ET epithelial cells. Since the ETs of guinea pig, mouse and rabbit are considerably smaller in diameter than the ET of pig, the surface forces in the ET may be more prominent and the surface-active agent may have different quality requirements in these small species.

The floor part of the ET lumen is postulated to be responsible for the mucociliary clearance functions (Bluestone 1996). In the lower airways, inhaled particles are transported in the liquid phase by mucociliary functions towards the pharynx. Surfactant is considered to be a primary immune barrier in the airways, changing the surfaces of particles and displacing them into liquid phases (Gehr et al. 1996). SP-A and SP-D expression and protein are detected in epithelial cells close to the mucosal folds in the floor part of the ET lumen. It is likely that these collectins participate in the local host defence system of ET. Similarly to the lower airways, SPs in ET may contribute to the non-specific defence in mucociliary clearance. Additionally, SPs may affect the specific defence system of ET by activating pro-inflammatory and anti-inflammatory effects, such as enhancing the phagocytosis of pathogens. Recently, evidence of SP-B influence on immune functions in lung has been presented (van Iwaarden et al. 2001). SP-B-containing vesicles could enhance the induction of immune responses via the airways. According to this concept, SP-B in ET may not affect surface tensions but could contribute to immune functions.

In lung, the collectins SP-A and SP-D have multiple functions both in surfactant homeostasis and in host defence (Crouch & Wright 2001). The postulated main role of SP-A and SP-D is to interact directly with carbohydrates on the surface of microbial pathogens and with surface receptors of inflammatory cells, thereby initiating a variety of effector mechanisms involved in the defence system. Especially in lung host defence, their functions overlap, as they both bind to and agglutinate the same pathogens. However, SP-A and SP-D also have complementary roles. In binding to pathogens, SP-A can utilize protein-protein or protein-lipid interactions in addition to sugar-protein binding, which is commonly utilized by SP-D (LPS binding). They also show different binding to influenza viruses. SP-D binds to glycoproteins in the virus membrane, whereas SP-A binds to viral neuraminidases with sialic acid residues in its carbohydrate recognition domain. Together, SP-A and SP-D may have a greater capacity to interact with pathogens and to enhance the function of phagocytic cells (Vaandrager & van Golde 2000). Similarly to the lower airways, SP-A and SP-D in the mucosal surface of ET could prevent the spreading of specific infections from the upper airways to the middle ear.

The long list of foreign substances that have been reported to bind to lung collectins include various bacteria, viruses, fungi and yeasts but also lipopolysaccharides (LPS, endotoxin) and allergens (Holmskov 2000). These micro-organisms further include

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Streptococcus pneumoniae and Haemophilus influenzae, which both cause respiratory infections and are the major bacteria causing middle ear infections (Bluestone et al. 1992). RSV and influenza viruses, i.e. the respiratory viruses that play an important role in the disruption of normal ET function, are also on the list of pathogens that SP-A and SP-D interact with (LeVine & Whitsett 2001).

Bronchiolar Clara cells contain electron-dense organelles that do not reveal multilamellated structures. Although SP-A, SP-D and SP-B have been localized in Clara cells, they do not otherwise morphologically resemble the cuboidal granular ET cells. Similarly, differences in the processing of SP-A and SP-D in Clara cells compared to alveolar type II epithelial cells have been reported (Mason et al. 1998). The functions of SPs in Clara cells, however, have not been clarified thus far. Studies with transgenic mice have shown a lack of processing of mature SP-B peptide in Clara cells, and the SP-B expressed under the Clara cell secretory protein promoter did not rescue SP-B-null mice (Lin et al. 1999). In the case of transgenic mice, however, the expression pattern may have been incomplete due to the abnormal promoter. Using IEM, secretion of SP-B from microvillar ET epithelial cells to the ET lumen could be detected here, suggesting complete processing of SP-B in ET epithelial cells. The immunoreactive SP-B protein detected in ETLF strongly resembles the SP-B dimer described as essential for surface tension reduction at the alveolar air/liquid interface (Beck et al. 2000a). The anti-sheep SP-B antibody recognized the 8 kDa mature SP-B in addition to the dimeric form.

The immunoreactivities for SP-A, SP-B and SP-D in ETLF were similar to those detected in BALF. Similarly to the alveolar surfactant (Froh et al. 1990), the aggregate fraction of ETLF contained immunoreactivities for SP-A and SP-B. In ETLF and in BALF, SP-A appeared as a typical protein triplet of 30-38 kDa bands, which are characteristic of reduced alveolar SP-A monomer. Both ETLF and BALF also had a 28-kDa band, which is the size of unglycosylated SP-A. The glycosylation of SP-A is thought to affect the immunological activity (van Iwaarden et al. 1992), and its different polymerisation could lead to different binding capacities with alterations of biological function (Hickling et al. 1998).

The non-sedimentable fraction of ETLF showed SP-D immunoreactivity similar to that of BALF. The 48-kDa SP-D was prominent. According to recent studies (van Eijk et al. 2000), porcine SP-D has three structural differences compared to other species. One of them is the extra cystein in the collagenous region. This could lead to different oligomerization and to a more stable heteromer. The other differences are the three amino-acid insertions and a potential extra N-glycosylation site in the CRD. Therefore, there may be size differences between porcine SP-D (48 kDa) and that of the other species studied so far (43 kDa).

According to previous morphologic evidence (Birken & Brookler 1972, Karchev et al. 1994, Bluestone 1996), the epithelial lining of the ET contains surface-active material, which possibly protects the epithelial lining and facilitates the muscle-driven opening of the ET during swallowing. This study, however, shows that the surface tension-lowering properties of ETLF are modest.

The distribution of unsaturated fatty acids in PC was distinctly higher in ETLF than in BALF. The distribution of PC16:0/16:0, the major surface-active phospholipid, was significantly lower in ETLF than in BALF and average when compared to other tissues. For a constant volume of recovered lavage fluid, the total PC concentration of ETLF was

55

one twentieth of that of BALF. The recovery of total PC per animal from ETLF was 1/10000 of the total PC recovered from BALF. The amount of total protein for a constant volume of recovered lavage fluid was higher in ETLF. While the different amounts of phospholipids may reflect the smaller surface area compared to the extensive alveolar surface of the lungs, it seems that when the total surface area of ET and lung are compared, the amounts may be somewhat similar. However, with these experiments, it is difficult to compare the amounts of SPs and the phospholipids in ET and in lung.

On the whole, however, since phospholipids, including some DPPC, were found in ETLF, it is possible that there is a specialized organelle or cell type involved in the secretion of such surfactant-like material. The similar phospholipid composition and molecular species between ETLF and ET epithelial cells serve as evidence to suggest that the material is secreted by these cells. SP-B was found to be secreted from the cuboidal epithelial cells that line ET. Analogous to alveolar surfactant, the phospholipid-containing material together with SPs in the epithelial lining of ET may be derived from the granular organelles of cuboidal ET cells.

As expected on the basis of the fatty acid structure of PC, ETLF was not surface-active. In measurements with a pulsating bubble surfactometer, surface tension revealed only small changes, and surface adsorption was instantaneous. This suggests that the aggregates from ETLF had detergent-like surface properties. The relatively high surface tension of material from ET is consistent with the notion that the ET remains closed unless the pharyngeal muscles force it to open. On the other hand, it is anticipated that the detergent-like properties of ETLF maintain the closure of the tube. Persistent patency is actually a factor that increases the risk of otitis (Bluestone 1985). Hence, the modest decrease in surface tension and the rapid surface spreading and adsorption tend to promote ET closure. Yet, the extracellular lining of the ET is likely to allow both solute transport and intermittent opening to occur without excessive shear forces or barotraumas. In the airways, the adhesive properties of mucus, which are influenced by its lipid composition and degree of hydration, are important in controlling the efficacy of mucus transport through ciliary activity (Gehr et al. 1996). In the ET, the detergent-like properties of the surface lining fluid may be crucial not only for the opening and closure of the tube but also for proper mucociliary clearance.

Appropriate ET function does not necessarily require a surface-active agent of pulmonary kind to maintain patency. In the alveoli and terminal airways, collapse is prevented by the surfactant phospholipids and SPs that enhance surface adsorption (Veldhuizen et al. 2000). Unlike the tidal gas delivery and gas exchange function of the lung, ET is involved in intermittent gas delivery into the middle ear cavity. ET must be virtually, if not totally, closed most of the time. The ventilatory function of ET is likely to be maintained by the surrounding muscles, which pull apart the mucous surfaces of the cartilaginous ET (Bluestone 1985). Intermittent swallowing, rather than regular breathing movements, accomplish the ventilatory function. The lumen of the tube is quite wide compared to the alveoli and the respiratory airways, and the muscles influence the opening of the tube. Thus, the mucociliary function of ET surfactant rather than its surface activity could be critical for proper functioning of the tube.

Lung surfactant has been proposed as a treatment of acute middle ear infections. White and his co-workers (1990) reported that surfactant phospholipids reduced the pressure required to force ET open in rats with ear infection. Venkatayan with his colleagues

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(2000) detected recently a similar effect with an intranasal aerosolized synthetic surfactant phospholipid preparation. The duration of infection was also decreased by the surfactant treatment. Permanent closure of ET is frequently evident in middle ear infections. Surface-active material is likely to be helpful in opening up the ET when its function is compromised by infection or inflammatory exudate that interferes with solute transport and tubal opening. Under these circumstances, the negative pressure generated in the middle ear cavity forces the exudate with its infectious content into the middle ear. In contrast to peripheral lung, where excess liquid may be removed across the alveolar epithelium, the liquid balance of the middle ear is critically dependent on the function of the ET. In order to prevent pressure-passive middle ear effusion, equilibration of pressures by intermittent ET opening is necessary (Monsell & Harley 1996). By removing the secretions and the excess liquid, the ciliary function complements the mechanical defence of the middle ear. However, despite the physical closure and particle removal mechanisms, the middle ear is exceptionally vulnerable to microbial attacks (Lim et al. 2000). According to this scheme, the absence of surfactant may not be the actual cause of infection, although we cannot exclude the possibility that primary dysfunction of ET surfactant may be responsible for an increased susceptibility to middle ear infection.

The pathogenesis of otitis media is very complicated, involving a network of interacting factors. The infection of the upper respiratory tract initiates this cascade of events, which finally leads to the development of acute otitis media as a complication. For otitis media to develop, microbes must colonize the nasopharynx and then enter the tubotympanum and replicate. This requires that both ET function, including ciliary function, and the host’s immune defence are disturbed (Heikkinen & Chonmaitree 2000). Spreading of the inflammation caused by respiratory pathogens contributes to the formation of exudate and the closure of the ET. The closure of the tube results in a negative pressure in the middle ear, which then leads to the suction of fluid, including inflammatory cells and microbes, into the middle ear cavity. The ET is the route for pathogens from the upper airways into the middle ear, and the lack of protecting agents in the ET mucosa may predispose infants to recurrent otitis media (Lim et al. 2000). According to this scheme, surfactant or surfactant proteins could be considered as a relevant therapy for preventing the spreading of inflammation by the pro- and anti-inflammatory properties of lung collectins. When the inflammation causes ET dysfunction and leads to tubal closure, surfactant may facilitate the opening of the tube and, thus, the removal of exudates.

Apart from protecting the lung, the collectins SP-A and SP-D are likely to play a crucial role in the local mucosal immunity of the ET. According to recent reports, moreover, SP-B in ET may also play a role in enhancing the immune functions. Similarly to the lower airways, SP-A and SP-D in ET may contribute to the host defence by trapping and aggregating pathogens for mucociliary clearance. SP-A and SP-D in the ET lumen may additionally promote the internalization of bacteria and viruses by phagocytic cells. The presence of SP-B but not surface-active phospholipids in ETLF suggests that the role of SP-B in ET differs from that in lung. Therefore, one might question the efficacy and rationale of a synthetic surfactant treatment in middle ear infections, as the preparations may be biologically incompatible. However, it would be interesting to

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consider the addition of SP-A or SP-D to the surfactant preparations to obtain a useful therapy for middle ear infections.

The specific functions of SPs in ET are an interesting target for further studies.

7 Conclusions

This study shows that the surfactant proteins A, B and D are expressed in the Eustachian tube and secreted into the ET lumen. SP-B, which was earlier regarded as lung-specific, seems to be present in other tissues as well.

The collectins SP-A and SP-D may act as immunomodulators affecting the clearance of pathogens. Similarly to the lower airways, SP-A and SP-D in the mucosal surface of the ET could prevent the spreading of specific infections from the upper airways to the middle ear. SP-A binds to and stimulates phagocytosis of the most common bacteria involved in middle ear infections. SP-D and SP-A also agglutinate viruses that associate with ear infections. The specific functions of SP-A and SP-D in the ET epithelium need to be studied further.

This study suggests that SP-B, expressed in specific cells in the ET epithelium and secreted from granules of cuboidal epithelial cells to the air/liquid lining of the ET, may improve mucociliary transport in the ET.

As a conclusion, the ET surfactant is different from the lung surfactant, and the low surface tension is not a major determinant of ETLF function. On the basis of current knowledge, surfactant proteins in ETLF are likely to be involved in the local host defense system of the ET and may contribute to the mucociliary function of the tube.

This study yielded further knowledge about the surfactant system in the ET. This could be important in understanding the mechanisms and agents involved in middle ear infections and, thus, in helping to manage and prevent this disease in the future.

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