Surfactant proteins of the human larynx

G Model AANAT-51055; No. of Pages 7

ARTICLE IN PRESS Annals of Anatomy xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Annals of Anatomy journal homepage: www.elsevier.com/locate/aanat

Surfactant proteins of the human larynx M. Sheats a , H. Schröder a , F. Rausch a , C. Bohr b , F. Kißlinger b , J. de Tristan b , H. Iro b , F. Garreis a , F. Paulsen a , M. Schicht a,1 , L. Bräuer a,∗,1 a b

Department of Anatomy II, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany Department of Otorhinolaryngology – Head & Neck Surgery, Friedrich-Alexander University Erlangen-Nürnberg, Erlangen, Germany

a r t i c l e

i n f o

Article history: Received 4 May 2016 Received in revised form 19 May 2016 Accepted 20 May 2016 Available online xxx Keywords: Surfactant proteins Larynx Epiglottis

a b s t r a c t Purpose: Surfactant proteins (SPs) originally identified in lung tissue are important players in the innate immune system. Beyond this, they contribute to stability and rheology of gaseous or aqueous interphases. In the present study, we determined the expression and presence of SPs (A, B, C and D) in different areas of the human larynx. Methods: mRNA expression of SP-A, -B, -C and -D was analyzed by means of RT-PCR in healthy samples of epiglottis, vocal and vestibular folds, subglottis and trachea. Distribution and localization of all four SPs were analyzed by Western blot and immunohistochemistry in healthy human tissue samples. Results: All four SPs were detected at the mRNA- and protein level in the human larynx as well as by means of immunohistochemistry in the different tissue samples of the human larynx. Conclusion: The results reveal that all four SPs are produced with different expression patterns within the human larynx. Based on the known functions, our results suggest that SPs might be involved in maintaining mucus rheology and subsequently they could be essential components for proper phonation. Moreover, the proteins seem to play a role in immune defense of the larynx. © 2016 Elsevier GmbH. All rights reserved.

1. Introduction The epithelium of the respiratory tract, including the tissues of the larynx, is lined by mucus (King et al., 1993; Leikauf et al., 1984). Particularly on the vocal folds, the mucus is a thin layer of liquid (Kawaida et al., 1990) serving as a physical and biochemical barrier protecting the underlying tissue from damage by inhaled particles and pathogens (Mogi et al., 1979). The surface covering liquid is necessary to maintain biomechanical characteristics of the vocal fold and, thus, it promotes normal voice quality (Sivasankar and Leydon, 2010). A further portion of the surface liquid is assumed to originate from secretions in the pulmonary airways and of laryngeal glands in the subglottis and supraglottis, especially in the laryngeal ventricles and the vestibular folds (Wanner, 1977). Surfactant is a complex mixture of different lipids and proteins lining the alveolar surface and reducing surface tension at the air-liquid interface of the lung, thereby preventing atelectasis

∗ Corresponding author at: Department of Anatomy II, University of Erlangen¨ Universitätsstraße 19, 91054 Erlangen, Germany. Nurnberg, Tel.: +49 9131 8526738; fax: +49 9131 8522862. E-mail address: [email protected] (L. Bräuer). 1 These authors contributed equally to the manuscript.

at the end of expiration (Lhert et al., 2007). The protein component is composed of the surfactant proteins A, B, C, D as well as the recently identified surfactant proteins G and H (Rausch et al., 2012; Schicht et al., 2014). While the surfactant’s phospholipid components are primarily responsible for lowering the alveolar surface tension, surfactant proteins play crucial roles in innate immune defense (El-Gendy et al., 2013). Surfactant proteins B and C are small, hydrophobic proteins with the main function of decreasing surface tension. They are critical to absorption of lipid molecules in the expanding phospholipid layer and thus enhance its stability during respiration. Therefore, both proteins are part of surfactant replacement therapies that treat respiratory distress syndrome in premature infants (El-Gendy et al., 2013). In addition, SP-B and SP-C are able to form proteolipid channels in phospholipid membranes, presumably playing an important role in ionic and lipid flows through the pulmonary surfactant membrane (Parra et al., 2013). SP-A and SP-D are proteins belonging to the collectin family, which is a subgroup of c-type lectins. C-tpye lectins play an important role in the immunologic protection of mucosal surfaces of the body. They interact directly with various bacteria, viruses, fungi and yeast by means of aggregation and opsonization. Furthermore, they suppress microbial growth indirectly by enhancing phagocytic uptake of pathogens by macrophages (Chroneos et al., 2010;

http://dx.doi.org/10.1016/j.aanat.2016.05.010 0940-9602/© 2016 Elsevier GmbH. All rights reserved.

Please cite this article in press as: Sheats, M., et al., Surfactant proteins of the human larynx. Ann. Anatomy (2016), http://dx.doi.org/10.1016/j.aanat.2016.05.010

G Model AANAT-51055; No. of Pages 7

ARTICLE IN PRESS M. Sheats et al. / Annals of Anatomy xxx (2016) xxx–xxx

2

Haagsman et al., 2008; Kuroki et al., 2007; Madsen et al., 2013; Wu et al., 2003). In addition, SP-A and D exhibit immune modulatory functions, either enhancing or reducing the immune response in order to maintain an inflammation free mucosal environment (Brandt et al., 2008; Lhert et al., 2007). The presence of surfactant proteins has meanwhile been demonstrated in a number of extrapulmonary tissues of the body, amongst them human nasal epithelium, digestive tract, mesentery, brain, lacrimal apparatus, gingiva and major salivary glands (Bourbon and Chailley-Heu, 2001; Bräuer et al., 2007a,b, 2012; Fisher and Mason, 1995; Schicht et al., 2013; Schob et al., 2013). The larynx is not only key to communication, but also a vitally important organ responsible for proper breathing and swallowing. Located at the crossroads between gastrointestinal and respiratory tract, the larynx faces distinct immunological challenges. It is continuously exposed to a variety of pollutants such as tobacco smoke, reflux, microbes and allergens (Barker et al., 2006). Being the narrowest point of the airway and the point of greatest air turbulence the deposition of inhaled particles is high (Thibeault et al., 2009). Despite the high incidence of acute and chronic inflammatory laryngeal diseases, rather little research about the larynx as an immunological organ has been published (Kutta et al., 2003). Recent studies suggest that the immune system of the larynx may serve as an immunological checkpoint not only influencing immune response in the larynx itself, but also in lung and gastrointestinal system (Thibeault et al., 2009). The vocal folds are covered by a thin layer of mucus (Bhattacharya and Siegmund, 2015; Sivasankar and Leydon, 2010). This layer functions as a physical and immunological barrier protecting the vocal folds from damage, pathogens and exsiccation. It contains carbohydrates, mucins and a variety of antimicrobial substances such as lactoferrin, lysozyme, and alpha and beta defensins (Kutta et al., 2002, 2007). Studies have shown that this mucus layer, depending on its viscosity, can influence vocal fold oscillation and thereby affect the quality of voice (Bonilha et al., 2012; Dollinger et al., 2014; Nakagawa et al., 1998). Up to now, there is no evidence that SP-A, SP-B, SP-C and SP-D are inherent proteins of the human larynx. In consideration of their biophysical and immunological properties we hypothesized that SPs could have great implications for the physiological function of the larynx. SPs could be important players in the epithelial antimicrobial defense as well as having impact on laryngeal mucus in regard to their rheological properties and, thus, on proper phonation. 2. Materials and methods 2.1. Tissues The study has been conducted in compliance with the institutional ethics committee, informed consent regulations, and the provisions of the Declaration of Helsinki. Tissue samples were obtained from 3 body donors (1 male, 2 female) who had donated their body to the Department of Anatomy II at the FriedrichAlexander University Erlangen-Nürnberg. The larynx of each body donor was dissected at least 24 h post mortem. Laryngeal tissue was taken from seven areas of each larynx. Regions of interest were epiglottis fat body, epiglottis (lingual side), epiglottis (laryngeal side), vestibular fold (inside the ventricle of Morgagni), vocal fold, subglottis and trachea. Half of each sample was immediately frozen in liquid nitrogen and stored at −80 ◦ C, the other half was fixed in a solution containing 4% paraformaldehyde. 2.2. Collection of mucus samples for ELISA quantification Mucus samples for ELISA were collected at the Department of Otorhinolaryngology, Head and Neck Surgery, Friedrich Alexander University Erlangen-Nürnberg, during diagnostic pan-endoscopy

Table 1 Sequences of primers used for RT-PCR analysis. Primer

Sequence

SP-A sense SP-A antisense SP-B sense SP-B antisense SP-C sense SP-C antisense SP-D sense SP-D antisense ␤-actin – sense ␤-actin – antisense

5 -GAT GGG CAG TGG AAT GAC AGG-3 5 -GGG AAT GAA GTG GCT AAG GGT G-3 5 -CAA ACG GCA TCT GTA TGC AC-3 5 -CGG AGA GAT CCT GTG TGT GA-3 5 -TCA TCG TCG TGG TGA TGG TG-3 5 -ATG GAG AAG GTG GCA GTG GTA A-3 5 -ATG TTG CTT CTC TGA GG-3 5 -TCA GAA CTC GCA-3 5 -TCA CCC ACA CTG TGC CCA TCT ACG A-3 5 -CAG CGG AAC CGC TCA TTG CCA ATG G-3

for various reasons (e.g. polyps, cysts, searching for benign or malignant tumors). To gain mucus samples of the larynx, patients were placed in a supine position with head extended and neck flexed (cf. Fig. 4). Subsequently a straight retractor (‘Kleinsasser´ıs Laryngoscope’) was placed and fixed in the supraglottal region. Due to the adhesive connection between the mucosa and the mucus layer, the endolarynx was rinsed with 10 ml saline (cf. Fig. 5). To ensure reaching the field of interest, the saline was irrigated with the help of an irrigation tube (cf. Fig. 6). This approach allowed for washing laryngeal mucus into the trachea. To avoid saline aspiration it was necessary to ensure that the cuff of the intubation tube was placed and insufflated correctly underneath the cricoid. Afterwards the saline could be suctioned and collected with the help of the irrigation tube and the 10 ml syringe. The acquired samples were transferred into sterile 1.5 ml reaction tubes and immediately frozen at −80 ◦ C. Subsequently, the specimens were thawed and centrifuged at 14,000 rpm for 10 min. The supernatants were transferred into a new 1.5 ml reaction tube and immediately frozen in liquid nitrogen and stored at −80 ◦ C for further use. 2.3. RNA preparation and synthesis of complementary cDNA RNA was isolated from the samples taken from different localizations of each larynx (see above) that were frozen and stored at −80 ◦ C using a Speed Mill Plus Homogenizer (Analytik Jena, Jena, Germany) and a Gene JET RNA purification kit (Thermo Scientific, Waltham, USA). Crude RNA was purified with isopropanol and repeated ethanol precipitation, and contaminating DNA was digested with RNase-free DNase I (30 min, 37 ◦ C; Fermentas/Thermo Scientific, Waltham, USA). Contamination of the purified RNA by genomic DNA was excluded by performing PCR with specific primers for SPs as well as for ␤-actin. In no case was amplification obtainable using the purified RNA as a template. The DNase was heat-denatured for 10 min at 75 ◦ C. A total amount of 2 ␮g RNA was used for each reaction: complementary DNA (cDNA) was generated with 1 ␮l Oligo(dT) (0.5 ␮g/100 pmol; Thermo Scientific, Waltham, USA) and 1 ␮l Revert Aid H Minus reverse transcriptase (200U Thermo Scientific, Waltham, USA) for 60 min at 37 ◦ C. The ubiquitously expressed ␤-actin which proved amplifiable in each case with the specific primer pair served as the internal control for cDNA integrity. 2.4. RT-PCR (reverse transcription) For conventional PCR, 2 ␮l cDNA (from each sample) was incubated with 9.8 ␮l H2 O, 2 ␮l 50 mM MgCl2 (Invitrogen, Carlsbad, USA), 2 ␮l dNTPs (10 mM), 2 ␮l 10× PCR buffer (Invitrogen, Carlsbad, USA), 0.2 ␮l (5U) Taq DNA-polymerase (Invitrogen, Carlsbad, USA), and 1 ␮l of each of the primers listed in Table 1. After 5 min of heat denaturation at 95 ◦ C, the PCR cycle consisted of 95 ◦ C for 30 s, 56.5 ◦ C (SP-A), 52 ◦ C (SP-B), 55.8 ◦ C (SP-C), or 57 ◦ C (SPD) for 30s each, and 72 ◦ C for 30 s. The number of PCR cycles

Please cite this article in press as: Sheats, M., et al., Surfactant proteins of the human larynx. Ann. Anatomy (2016), http://dx.doi.org/10.1016/j.aanat.2016.05.010

G Model AANAT-51055; No. of Pages 7

ARTICLE IN PRESS M. Sheats et al. / Annals of Anatomy xxx (2016) xxx–xxx

3

Table 2 Primary antibodies used for Western blot analysis and immunohistochemistry. Antibody

Method

Dilution

Company, Catalog number

Mouse anti human SP-A Mouse anti human SP-B Rabbit anti human SP-B Rabbit anti human SP-C Mouse anti human SP-D Rabbit anti human SP-D

IHC, WB IHC, WB IHC, WB IHC, WB IHC, WB IHC, WB

1:500 1:500 1:500 1:500 1:500 1:500

Millipore; MAB3270 Millipore; MAB 3276 Abcam, AB40876 Millipore, MAB3786 Acris, BM4083 Millipore, AB3434

used added up for 39 for each primer pair. The final elongation step consisted of 72 ◦ C for 4 min. The primers were synthesized by MWG-Biotech AG (Ebersberg, Germany). Ten microliters of the PCR product were loaded on an agarose gel after addition of 5 ␮l loading dye. After electrophoresis, the amplified products were visualized by means of fluorescence in a dark hood. The size of each obtained PCR product in base-pairs (bp) was compared with the expected size for each surfactant protein mRNA. For verification and comparison, bacterial plasmids carrying open reading frames (ORF) for the investigated proteins were used as a reference (German Resource Centre for Genome Research GmbH; SP-A, IRAUp969H0686D6; SP-B, IRAKp961K1368Q2; SP-C, IKAUp969F0244D6; SP-D, IRAUp969D0386D6). 2.5. Antibodies Antibodies listed in Table 2 were used for Western blot analysis as well as for immunohistochemical investigations as specified by the manufacturer. 2.6. Western blot Tissue samples obtained from the laryngeal regions mentioned above were homogenized in 1% (v/v) Triton X-100 buffer on ice with a tissue homogenizer (IKAT 10 Basic Ultra Turrax Homogenizer, Cole Parmer). After homogenization the samples were incubated on ice for 30 minutes and subsequently centrifuged at 13,000 × g for 20 min. The supernatant was transferred to a new 1.5 ml reaction tube and used for further analysis. Protein concentration was determined via Bradford assay. Total protein (30 ␮g) was then analyzed by Western blot. Proteins were resolved by reducing 15% SDS-polyacrylamide gel electrophoresis, electrophoretically transferred at room temperature for 2 h at 0.8 mA/cm2 onto a 0.45 ␮m pore size nitrocellulose membrane. Bands were detected with primary antibodies listed in Table 2 and secondary antibodies (anti-rabbit/anti-mouse IgG, respectively, conjugated to horseradish peroxidase, 1:5000, cf. Table 3) applying chemiluminescence (ECL-Plus; Amersham-Pharmacia, Uppsala, Sweden). Human lung protein was used as positive control for antibody reactivity. Molecular weights of the detected protein bands were estimated using standard proteins (Prestained Protein Ladder, Fermentas, St. Leon-Rot, Germany) ranging from 11 to 170 kDa. 2.7. Immunohistochemistry For immunohistochemistry, paraformaldehyde fixed tissue specimens from the epiglottis, vestibular folds, vocal folds, and trachea were embedded in paraffin, sectioned (6 ␮m) and dewaxed. Table 3 Secondary antibodies used for Western blot analysis and immuno-histochemistry. Antibody

Company, Catalog number

Anti-rabbit IgG, HRP conjugated Anti-mouse IgG, HRP conjugated Anti-rabbit IgG, biotinylated

Dako, P0448 Dianova Dako, P0432

Immunohistochemical staining was performed with polyclonal antibodies directed against the four SPs (cf. Table 2). For antigen retrieval, specimens were boiled in citrate buffer (pH 6) for 10 min. Non-specific binding was inhibited by incubation with rabbit normal serum (SP-A) or goat normal serum (SP-B, SP-C, SP-D) (Dako GmbH, Hamburg, Germany) 1:5 in Tris-buffered saline (TBS). Each primary antibody (1:50) was applied overnight at 4 ◦ C. The secondary antibodies (1:100) were incubated at room temperature for 1 h. Visualization was achieved with aminoethylcarbazole (AEC) for at least 5 min. Red stained areas within the tissues indicate a positive antibody reaction. After counterstaining with hemalum, the sections were mounted in Aquatex (Aquatex; Roche Applied Science, Basel, Switzerland). Two negative control sections were used in each case: one was incubated with the secondary antibody only, and the other one with the primary antibody only. All slides were examined with a Keyence Biorevo BZ9000 microscope. 2.8. ELISA Commercially available enzyme-linked immunosorbent assay kits (Cusabio, Wuhan, China) were used to quantify the amount of SP-A (CSB-E08683h), SP-B (CSB-E10134h), SP-C (CSB-E10135h) and SP-D (CSB-E11166h) in laryngeal lavages. The analysis was performed using a microplate spectrophotometer at a wavelength of 450 nm and 405 nm for measuring the absorbance. Values for antigen concentration were determined by comparison with a standard dilution series provided by Cusabio. Each sample was calculated as surfactant protein in ng/total protein in mg (ng/mg). 3. Results 3.1. Region of the larynx Tissue from the following regions of the larynx was investigated by RT-PCR and Western blot analysis: lingual epiglottis, laryngeal epiglottis, vestibular fold, vocal fold, subglottic region and trachea. Tissue from epiglottis fat body was used as local expression control without epithelial function. 3.2. RT-PCR RT-PCR was performed in order to verify that surfactant protein A, B, C and D mRNA is transcribed by the different sections of the larynx (cf. Fig. 1). Specific amplification products for all investigated surfactant proteins could be detected within the regions mentioned above in all larynges examined (n = 3), (SP-A: 121 bp, SP-B: 194 bp, SP-C: 110 bp, SP-D: 461 bp). The size of each amplification product was equivalent to the expected PCR products. ␤-actin PCR (positive control for cDNA synthesis) revealed distinct bands at 298 bp as expected. 3.3. Western blot Protein isolates from the different samples of 3 larynges were tested for the presence of SP-A, SP-B, SP-C and SP-D (cf. Fig. 2). Distinct bands could be detected for SP-A at 60 kDa and 28 kDa,

Please cite this article in press as: Sheats, M., et al., Surfactant proteins of the human larynx. Ann. Anatomy (2016), http://dx.doi.org/10.1016/j.aanat.2016.05.010

G Model AANAT-51055; No. of Pages 7

ARTICLE IN PRESS M. Sheats et al. / Annals of Anatomy xxx (2016) xxx–xxx

4

Fig. 1. RT-PCR analysis of surfactant protein mRNA from samples of different laryngeal areas. Negative control was performed without template. As positive control, a plasmid carrying the open reading frame (ORF) for the respective SPs was used. All RT-PCR analyses show cDNA amplification for the relevant surfactant protein in comparison to ␤-actin.

SP-B at 40 kDa, SP-C at 12 kDa and 21 kDa and SP-D at 43 kDa for each of the specimens. Only in case of SP-C the detected bands were blurry resulting from the hydrophobicity of the protein. A protein isolate from lung tissue served as positive control and was treated and incubated under the same conditions. The positive control showed distinct bands at the same molecular weights as the examined specimens of the larynges. 3.4. Immunohistochemistry Sections from lingual as well as laryngeal epiglottis, vestibular fold, vestibular fold glands and trachea were investigated by means of immunohistochemistry. Negative control (secondary antibody only) was conducted for each of the investigated tissues and remained unstained (cf. Fig. 3). SP-A could be localized diffusely distributed in the cytoplasm of basal and squamous cells of the stratified non-keratinized epithelium on the lingual side of the epiglottis. Additionally, in squamous epithelial cells more intensely stained granular structures were

detectable. In the ciliated pseudostratified columnar epithelium of the laryngeal side SP-A was mainly present in the upper epithelial layers both diffusely distributed in the cytoplasm as well as localized in granular structures. Similar to that the vestibular fold revealed diffuse cytoplasmic reactivity as well as granular reactivity in all areas of the epithelium with particularly stronger reactivity in the uppermost epithelial layer. In the glands of the vestibular fold, serous as well as mucous cells displayed positive immunoreactivity for SP-A. In the vestibular fold, SP-A was present in all layers of the epithelium. The strongest reactivity was visible paracellularly in the upper layers. Tracheal epithelium revealed a similar distribution pattern with diffuse and granular antibody reactivity in the superficial layers (cf. Fig. 3A). SP-B revealed an overall weaker immunoreactivity compared to the other surfactant proteins. On the lingual side of the epiglottis, isolated single squamous cells presented precipitations of SP-B. On the laryngeal side, SP-B was only present in the superficial cell layers. SP-B displayed a subjectively stronger immunoreactivity in the vestibular fold, especially in the ciliated apical cells. Additionally,

Fig. 2. Western blot analysis of samples from different areas of the human larynx and trachea using anti-SP-antibodies. Lung tissue served as positive control, whereas GAPDH was used as loading control at 37 kDa.

Please cite this article in press as: Sheats, M., et al., Surfactant proteins of the human larynx. Ann. Anatomy (2016), http://dx.doi.org/10.1016/j.aanat.2016.05.010

G Model AANAT-51055; No. of Pages 7

ARTICLE IN PRESS M. Sheats et al. / Annals of Anatomy xxx (2016) xxx–xxx

5

Fig. 3. Immunohistochemical detection of surfactant proteins in different areas of the human larynx and from trachea. All investigated samples show immunoreactivity (red staining) to a differing extent mostly in the superficial epithelial layer as well as in the mucous areas of the vestibular fold glands. Insets show magnifications of the respective tissue. Scale bars 100 ␮m.

SP-B was present in granular form throughout the epithelium. In the glands of the vestibular fold, only faint cytoplasmatic signals could be detected in a few cells. In the vestibular fold, SP-B was expressed by the most apical cell layer of the non-stratified squamous epithelium. In addition, predominantly cells located in the basal cell layers presented perinuclear reactivity. In tracheal epithelium, SP-B was located diffusely in the upper parts of the epithelium (cf. Fig. 3B). SP-C was primarily located in the squamous and basal cell layers in the stratified squamous epithelium of the lingual side of the epiglottis. Here SP-C revealed cytoplasmatic and a more intense perinuclear immunoreactivity. The uppermost cell layer presented a stronger reactivity. On the laryngeal side, we detected a diffuse staining in the upper cell layers and granular staining throughout the epithelium. In a comparable manner the epithelium of the vestibular fold presented a diffuse and granular staining with intense reactivity in the ciliated cells. The glands were less strongly stained, presenting only light cytoplasmatic reactivity and scattered cells with SP-C in granular form. In the vestibular fold, SP-C was located mainly in the cytoplasma und perinuclearly in the lower and basal cell layers. In tracheal epithelium, SP-C revealed a stronger immunoreactivity in the upper cell layers, the more basal layers show diffuse and granular reactivity (cf. Fig. 3C). SP-D subjectively showed an overall strong immunoreactivity. The stratified squamous epithelium of the epiglottis displayed intense immunoreactivity in the squamous cells. SP-D was diffusely located in the cytoplasm, forming precipitations within the cell and furthermore showing paracellular reactivity. In addition to that, SP-D revealed immunoreactivity in the very basal cells of this epithelium. On the laryngeal side, SP-D was mostly present in the upper layers, staining cytoplasm of the cells and forming precipitations. In the vestibular fold, SP-D was homogenously distributed throughout the epithelium, individual cells revealed a strong apical accumulation of SP-D. In the glands of the vestibular fold, SP-D was

found in granules of different size and diffusely in the cytoplasm of serous as well as mucous cells. In the epithelium of the vestibular fold, SP-D displayed homogenous immunoreactivity. It was present in cytoplasm as well as paracellularly. In the trachea, cytoplasmic immunoreactivity was evenly distributed in the epithelium. Furthermore SP-D formed disseminated precipitations (cf. Fig. 3D). 3.5. ELISA Laryngeal lavages from 20 patients were investigated (5 patients undergoing routine otolaryngological inspection without any laryngeal carcinoma or alteration and 15 patients suffering from laryngeal squamous cell carcinoma). Unfortunately, the measurement as well as the statistical analysis of the surfactant protein

Fig. 4. Patient preparation and intraoperative setup for laryngeal lavage during direct laryngosopy in an intubated patient during general anaesthesia.

Please cite this article in press as: Sheats, M., et al., Surfactant proteins of the human larynx. Ann. Anatomy (2016), http://dx.doi.org/10.1016/j.aanat.2016.05.010

G Model AANAT-51055; No. of Pages 7

ARTICLE IN PRESS M. Sheats et al. / Annals of Anatomy xxx (2016) xxx–xxx

6

Fig. 5. Laryngeal lavage with 10 cc saline under direct lanrygoscopy.

Fig. 6. Laryngeal lavage under direct laryngoscopy in a patient with a tumour of the right vocal fold.

expression could not be analyzed, because the protein concentration of the samples was too scattered and beyond the detectable range for the ELISA. 4. Discussion The results of this study clearly demonstrate that surfactant proteins A-D are inherent components of laryngeal mucosa. All PCR products correspond with the expected length as well as protein translation products detected by means of Western blot analysis (SP-A: 28 kDa, 60 kDa; SP-B: 8 kDa, 40 kDa; SP-C: 12 kDa, 21 kDa; SP-D: 43 kDa). In case of SP-A the band at 28kDA represents the mature monomeric isoform, the band at 60 kDa the dimeric form of SP-A (Floros, 2001). For SP-B and SP-C the antibody detected premature forms of the proteins at 40 kDa and 21 kDa (Voorhout et al., 1992). It has been widely reported that surfactant proteins undergo post-translational modifications such as phosphorylation, glycosylation, palmitoylation and formation of disulphide bonds (Floros, 2001; Voorhout et al., 1992; Whitsett et al., 1986). These modifications allow the oligomerization of proteins on the one hand and a shift in physicochemical properties (amphiphilic) on the other. Both lead to different molecular patterns and functions. In this context, several studies suggest that multimerization of SP-A and SP-D is crucial for influenza-A virus neutralization, opsonization of Pneumocytis carinii and inhibition of LPS induced cytokine production

(Atochina-Vasserman et al., 2010). In the case of SP-C Western blot analysis, the detected protein bands are blurry, which is well known to be a result of the strong hydrophobicity of the protein. Immunohistochemistry revealed that SP-A and SP-D are produced in most cells of the investigated epithelia and also in the seromucous glands of the vestibular fold. With regard to their functions in other tissues, SP-A and SP-D probably contribute to the innate mucosal immune defense. Both are well known to be pattern recognition molecules with lectin domains that bind to sugars on microbial surfaces (Sorensen et al., 2007). Furthermore, SPs are able to directly initiate cell membrane lysis of gram negative bacteria as well as inhibit fungal growth (Sorensen et al., 2007). At the same time, SP-A and SP-D can either attenuate or enhance inflammatory responses in a microbial ligand specific manner to secure sufficient preservation of inflammation while averting damage to host tissue of the glottis (Haagsman et al., 2008). Up to now, a number of other antimicrobial substances and antimicrobial peptides such as lactoferrin, lysozyme, alpha and beta defensins and others have been detected in the larynx (Kutta et al., 2003). These proteins have features that are also typical of SP-A and SP-D like bacteriostatic and bactericidal effects and are well known as ingredients of the surface liquid lining the alveoli (Kutta et al., 2002, 2007). Considering the similar immunological functions that the two systems are facing as well as the similarity in innate host defense of the molecules we assume that SP-D and SP-A are also important players in innate immunity in the larynx. The results of the immunohistochemical investigations reveal that SP-B and SP-C are mainly produced by cells of the superficial layer of the examined epithelia whereas the seromucous glands of the vestibular folds showed weaker reactivity. In this context, and with regard to their function, the surfactant proteins B and C might be essential to the formation and stability of a mucus layer due to their ability to reduce surface tension at the air liquid interface surrounding the vocal folds in the larynx, thus facilitating mucus rheology and laryngeal secretion. Similar findings were observed in the epithelium of the Eustachian tube and in nasal epithelium (McGuire, 2002; Schicht et al., 2013). SPB plays a crucial role during surfactant homeostasis by facilitating the adsorption of lipid molecules into the expanding monolayer. Moreover, it regulates the viscosity of phospholipid layers. We hypothesize that the surface active and rheological properties of SP-B and SP-C could influence the laryngeal mucus layer. In this context, Kutta et al. (2004) already demonstrated that trefoil factor family (TFF) peptides might play a role in the viscous function of mucus secreted onto the vocal folds and thus are important components of voice production. Several studies suggest that this mucus layer, depending on its surface tension and viscosity, can influence the aerodynamics and mechanical vibration of the vocal folds and, thus, either enhances or lowers the quality of voice (Bonilha et al., 2012; Dollinger et al., 2014; Nakagawa et al., 1998). Moreover, an intact fluid film and mucociliary transport function is crucial to self-cleaning and natural defense of the laryngeal mucosa. Dysregulation of this system, e.g. in case of laryngitis and malignant or inflammatory processes, may result in negative changes in the moisture balance and the consistency of the laryngeal mucus, thus leading to hoarseness and functional dysphonia (Ayache et al., 2004). To estimate and standardize the distribution and concentration of the surfactant proteins of the vocal fold, we established a method based on pan-endoscopy using ‘Kleinsasser’s Laryngoscope’ (cf. Section 2) to obtain laryngeal mucus and lavages. Unfortunately, this method seems to be unsuitable for our approach because the collected protein samples strongly differ with regard to the mean protein concentration. Furthermore, in most of the lavages, the concentration of the surfactant proteins was beneath the detection level of the ELISA assay. Brush biopsy or tissue biopsies could be an

Please cite this article in press as: Sheats, M., et al., Surfactant proteins of the human larynx. Ann. Anatomy (2016), http://dx.doi.org/10.1016/j.aanat.2016.05.010

G Model AANAT-51055; No. of Pages 7

ARTICLE IN PRESS M. Sheats et al. / Annals of Anatomy xxx (2016) xxx–xxx

alternative method to determine the levels of protein expression, which needs further elucidation. In conclusion, our results demonstrate that surfactant proteins seem to be essential components of the larynx – necessary for optimal mucus rheology and thus proper phonation on the one hand and essential to a stable innate local immune system on the other hand. Acknowledgments This work was supported in part by the Deutsche Forschungsgemeinschaft (DFG, Program Grants BR 1329/12-1 and BR 3681/2-1). The authors thank Anke Fischer-Gößwein, Jessica Braun, Maike Hemmerlein and Hong Nguyen for excellent technical assistance. References Atochina-Vasserman, E.N., Beers, M.F., Gow, A.J., 2010. Review: chemical and structural modifications of pulmonary collectins and their functional consequences. Innate Immun. 16, 175–182. Ayache, S., Ouaknine, M., Dejonkere, P., Prindere, P., Giovanni, A., 2004. Experimental study of the effects of surface mucus viscosity on the glottic cycle. J. Voice 18, 107–115. Barker, E., Haverson, K., Stokes, C.R., Birchall, M., Bailey, M., 2006. The larynx as an immunological organ: immunological architecture in the pig as a large animal model. Clin. Exp. Immunol. 143, 6–14. Bhattacharya, P., Siegmund, T., 2015. The role of glottal surface adhesion on vocal folds biomechanics. Biomech. Model Mechanobiol. 14, 283–295. Bonilha, H.S., White, L., Kuckhahn, K., Gerlach, T.T., Deliyski, D.D., 2012. Vocal fold mucus aggregation in persons with voice disorders. J. Commun. Disord. 45, 304–311. Bourbon, J.R., Chailley-Heu, B., 2001. Surfactant proteins in the digestive tract, mesentery, and other organs: evolutionary significance. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 129, 151–161. Brandt, E.B., Mingler, M.K., Stevenson, M.D., Wang, N., Khurana Hershey, G.K., Whitsett, J.A., Rothenberg, M.E., 2008. Surfactant protein D alters allergic lung responses in mice and human subjects. J. Allergy Clin. Immunol. 121, 1140–1147, e1142. Bräuer, L., Johl, M., Borgermann, J., Pleyer, U., Tsokos, M., Paulsen, F.P., 2007a. Detection and localization of the hydrophobic surfactant proteins B and C in human tear fluid and the human lacrimal system. Curr. Eye Res. 32, 931–938. Bräuer, L., Kindler, C., Jager, K., Sel, S., Nolle, B., Pleyer, U., Ochs, M., Paulsen, F.P., 2007b. Detection of surfactant proteins A and D in human tear fluid and the human lacrimal system. Invest. Ophthalmol. Vis. Sci. 48, 3945–3953. Bräuer, L., Schicht, M., Stengl, C., Heinemann, F., Gotz, W., Scholz, M., Paulsen, F., 2012. Detection of surfactant proteins A, B, C, and D in human gingiva and saliva. Biomed. Tech. (Berl) 57, 59–64. Chroneos, Z.C., Sever-Chroneos, Z., Shepherd, V.L., 2010. Pulmonary surfactant: an immunological perspective. Cell. Physiol. Biochem. 25, 13–26. Dollinger, M., Grohn, F., Berry, D.A., Eysholdt, U., Luegmair, G., 2014. Preliminary results on the influence of engineered artificial mucus layer on phonation. J. Speech Lang. Hear. Res. 57, S637–S647. El-Gendy, N., Kaviratna, A., Berkland, C., Dhar, P., 2013. Delivery and performance of surfactant replacement therapies to treat pulmonary disorders. Ther. Deliv. 4, 951–980. Fisher, J.H., Mason, R., 1995. Expression of pulmonary surfactant protein D in rat gastric mucosa. Am. J. Resp. Cell Mol. Biol. 12, 13–18. Floros, J., 2001. Human surfactant protein A (SP-A) variants: why so many, why such a complexity? Swiss Med. Wkly. 131, 87–90. Haagsman, H.P., Hogenkamp, A., van Eijk, M., Veldhuizen, E.J., 2008. Surfactant collectins and innate immunity. Neonatology 93, 288–294. Kawaida, M., Fukuda, H., Kano, S., Shiotani, A., Kohno, N., 1990. Dynamic movement of air tract fluid in lubrication of the larynx during phonation: a basic study using excised canine larynges and experimental air tract fluid by means of X-ray stroboscope system. Auris Nasus Larynx 16, 237–243.

7

King, M., Agarwal, M., Shukla, J.B., 1993. A planar model for mucociliary transport: effect of mucus viscoelasticity. Biorheology 30, 49–61. Kuroki, Y., Takahashi, M., Nishitani, C., 2007. Pulmonary collectins in innate immunity of the lung. Cell. Microbiol. 9, 1871–1879. Kutta, H., Knipping, S., Claassen, H., Paulsen, F., 2007. Functional anatomy of the larynx from clinical points of view: part II: Laryngeal mucous membrane, blood supply, innervation, lymphatic drainage, age-related changes. HNO 55, 661–675, quiz 676. Kutta, H., Steven, P., Kohla, G., Tillmann, B., Paulsen, F., 2002. The human false vocal folds – an analysis of antimicrobial defense mechanisms. Anat. Embryol. (Berl) 205, 315–323. Kutta, H., Steven, P., Tillmann, B.N., Tsokos, M., Paulsen, F.P., 2003. Region-specific immunological response of the different laryngeal compartments: significance of larynx-associated lymphoid tissue. Cell Tissue Res. 311, 365–371. Kutta, H., Steven, P., Varoga, D., Paulsen, F.P., 2004. TFF peptides in the human false vocal folds of the larynx. Peptides 25, 811–818. Leikauf, G.D., Spektor, D.M., Albert, R.E., Lippmann, M., 1984. Dose-dependent effects of submicrometer sulfuric acid aerosol on particle clearance from ciliated human lung airways. Am. Ind. Hyg. Assoc. J. 45, 285–292. Lhert, F., Yan, W., Biswas, S.C., Hall, S.B., 2007. Effects of hydrophobic surfactant proteins on collapse of pulmonary surfactant monolayers. Biophys. J. 93, 4237–4243. Madsen, J., Gaiha, G.D., Palaniyar, N., Dong, T., Mitchell, D.A., Clark, H.W., 2013. Surfactant protein D modulates HIV infection of both T-cells and dendritic cells. PLoS ONE 8, e59047. McGuire, J.F., 2002. Surfactant in the middle ear and eustachian tube: a review. Int. J. Pediatr. Otorhinolaryngol. 66, 1–15. Mogi, G., Watanabe, N., Maeda, S., Umehara, T., 1979. Laryngeal secretions. An immunochemical and immunohistological study. Acta Otolaryngol. 87, 129–141. Nakagawa, H., Fukuda, H., Kawaida, M., Shiotani, A., Kanzaki, J., 1998. Lubrication mechanism of the larynx during phonation: an experiment in excised canine larynges. Folia Phoniatr. Logop. 50, 183–194. Parra, E., Alcaraz, A., Cruz, A., Aguilella, V.M., Perez-Gil, J., 2013. Hydrophobic pulmonary surfactant proteins SP-B and SP-C induce pore formation in planar lipid membranes: evidence for proteolipid pores. Biophys. J. 104, 146–155. Rausch, F., Schicht, M., Paulsen, F., Ngueya, I., Brauer, L., Brandt, W., 2012. SP-G, a putative new surfactant protein – tissue localization and 3D structure. PLoS ONE 7, e47789. Schicht, M., Knipping, S., Hirt, R., Beileke, S., Sel, S., Paulsen, F., Brauer, L., 2013. Detection of surfactant proteins A, B, C, and D in human nasal mucosa and their regulation in chronic rhinosinusitis with polyps. Am. J. Rhinol. Allergy 27, 24–29. Schicht, M., Rausch, F., Finotto, S., Mathews, M., Mattil, A., Schubert, M., Koch, B., Traxdorf, M., Bohr, C., Worlitzsch, D., Brandt, W., Garreis, F., Sel, S., Paulsen, F., Brauer, L., 2014. SFTA3, a novel protein of the lung: three-dimensional structure, characterisation and immune activation. Eur. Respir. J. 44, 447–456. Schob, S., Schicht, M., Sel, S., Stiller, D., Kekule, A.S., Paulsen, F., Maronde, E., Brauer, L., 2013. The detection of surfactant proteins A, B, C and D in the human brain and their regulation in cerebral infarction, autoimmune conditions and infections of the CNS. PLoS ONE 8, e74412. Sivasankar, M., Leydon, C., 2010. The role of hydration in vocal fold physiology. Curr. Opin. Otolaryngol. Head Neck Surg. 18, 171–175. Sorensen, G.L., Husby, S., Holmskov, U., 2007. Surfactant protein A and surfactant protein D variation in pulmonary disease. Immunobiology 212, 381–416. Thibeault, S.L., Rees, L., Pazmany, L., Birchall, M.A., 2009. At the crossroads: mucosal immunology of the larynx. Mucosal Immunol. 2, 122–128. Voorhout, W.F., Veenendaal, T., Haagsman, H.P., Weaver, T.E., Whitsett, J.A., van Golde, L.M., Geuze, H.J., 1992. Intracellular processing of pulmonary surfactant protein B in an endosomal/lysosomal compartment. Am. J. Physiol. 263, L479–L486. Wanner, A., 1977. Clinical aspects of mucociliary transport. Am. Rev. Respir. Dis. 116, 73–125. Whitsett, J.A., Hull, W.M., Ohning, B., Ross, G., Weaver, T.E., 1986. Immunologic identification of a pulmonary surfactant-associated protein of molecular weight = 6000 daltons. Pediatr. Res. 20, 744–749. Wu, H., Kuzmenko, A., Wan, S., Schaffer, L., Weiss, A., Fisher, J.H., Kim, K.S., McCormack, F.X., 2003. Surfactant proteins A and D inhibit the growth of Gramnegative bacteria by increasing membrane permeability. J. Clin. Invest. 111, 1589–1602.

Please cite this article in press as: Sheats, M., et al., Surfactant proteins of the human larynx. Ann. Anatomy (2016), http://dx.doi.org/10.1016/j.aanat.2016.05.010