Nitric Oxide 44 (2015) 3–7
Contents lists available at ScienceDirect
Nitric Oxide j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y n i o x
Brief Communication
Neuronal NOS localises to human airway cilia Claire L. Jackson a,b,*, Jane S. Lucas a,b, Woolf T. Walker a,b, Holly Owen a, Irnthu Premadeva a, Peter M. Lackie a,b a
Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK Primary Ciliary Dyskinesia Centre, NIHR Southampton Respiratory Biomedical Research Unit, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK b
A R T I C L E
I N F O
Article history: Received 25 June 2014 Revised 21 October 2014 Available online 6 November 2014 Keywords: NOS EC 1.14.13.39 nNOS Cilia Airway Epithelium
A B S T R A C T
Background: Airway NO synthase (NOS) isoenzymes are responsible for rapid and localised nitric oxide (NO) production and are expressed in airway epithelium. We sought to determine the localisation of neuronal NOS (nNOS) in airway epithelium due to the paucity of evidence. Methods and results: Sections of healthy human bronchial tissue in glycol methacrylate resin and human nasal polyps in paraffin wax were immunohistochemically labelled and reproducibly demonstrated nNOS immunoreactivity, particularly at the proximal portion of cilia; this immunoreactivity was blocked by a specific nNOS peptide fragment. Healthy human epithelial cells differentiated at an air–liquid interface (ALI) confirmed the presence of all three NOS isoenzymes by immunofluorescence labelling. Only nNOS immunoreactivity was specific to the ciliary axonemeand co-localised with the cilia marker β-tubulin in the proximal part of the ciliary axoneme. Conclusions: We report a novel localisation of nNOS at the proximal portion of cilia in airway epithelium and conclude that its independent and local regulation of NO levels is crucial for normal cilia function. © 2014 Elsevier Inc. All rights reserved.
1. Background Nitric oxide (NO) is a mobile, reactive and ubiquitous signalling molecule within the airway, regulating vascular and bronchial tone, airway permeability [1] and ciliary beat frequency (CBF) [2–5]. It is pro-inflammatory with anti-bacterial action [6,7] and tumouricidal activity [8]. NO is generated from L-arginine by the catalytic activity of nitric oxide synthase (NOS) isoenzymes. The NOS isoenzymes, encoded by NOS1–3 genes on different chromosomes, include neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) respectively. NOS activity is dependent on the presence of cofactors including nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide, and heme [6,9–12].
NADPH reactivity, iNOS and eNOS are localised in the epithelial cell layer of the healthy and allergic human nasal mucosa [5,13–16]. Bronchial epithelium biopsied from atopic asthmatic subjects demonstrated immunoreactivity for nNOS, iNOS and eNOS throughout the columnar and basal cells, with prominent staining for each at the apical surface of columnar cells [17]. Neuronal NOS is also reported in nervous, vascular, gut epithelium but there is little data from nasal or bronchial epithelium, with no reports of localisation in cilia [14,16–19]. The aim of our study was to determine the expression of nNOS protein in healthy human airway ciliated epithelium. 2. Methods 2.1. Ethics statement
Abbreviations: ALI, air–liquid interface; BEGM, bronchial epithelial cell growth medium; CBF, ciliary beat frequency; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; DAB, 3,3′-diaminobenzidine; GMA, glycol methacrylate; IFT, intraflagella transport; IgG, immunoglobulin G; IL-1β, interleukin-1 beta; NADPH, nicotinamide adenine dinucleotide phosphate; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NOS, nitric oxide synthase; NOS1–3, neuronal nitric oxide synthase 1–3; PBS, phosphate-buffered saline; PKA, protein kinase A; PKIs, protein kinase A inhibitors; PIN, protein inhibitors of neuronal nitric oxide synthase; TBS, tris-buffered saline. * Corresponding author. Clinical and Experimental Sciences, University of Southampton Faculty of Medicine, Southampton General Hospital, Mailpoint 12, Level B South Block, Tremona Road, Southampton SO16 6YD, UK. Fax: 023 8120 5230. E-mail address: [email protected] (C.L. Jackson). http://dx.doi.org/10.1016/j.niox.2014.11.003 1089-8603/© 2014 Elsevier Inc. All rights reserved.
Our research received approval from the National Research Ethics Service (NRES), South Central Committee (06/Q1702/109) and locally at the University Hospital Southampton NHS Foundation Trust Research and Development Department (RHMCH10395). All subjects gave written informed consent. 2.2. Immunohistochemical labelling of nitric oxide synthases Human nasal polyp (n = 7 subjects) in paraffin wax sections and healthy human bronchial tissue (n = 3 subjects) in glycol
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C.L. Jackson et al./Nitric Oxide 44 (2015) 3–7
methacrylate (GMA) resin sections were immunostained using standard protocols. For paraffin wax sections, antibodies were applied following heat based antigen retrieval in 10 mM citrate buffer (pH 6.0). The Calbiochem (Merck, UK) primary rabbit polyclonal antinNOS (1414–1434) antibody (1 mg/ml stock) was used at 1:1500 for paraffin wax sections and 1:100 for nNOS in GMA resin sections and incubated overnight at 4 °C. A primary mouse monoclonal β-tubulin antibody (Sigma) (ascites fluid) used at 1:15,000 was used as a positive control for cilia staining in GMA resin sections. Equivalent concentrations of an anti-IgG isotype antibody were used as a negative control. The synthetic peptide fragment CRSESIAFIEESKKDTDEVFSS corresponding to amino acids 1414– 1434 of nNOS (4 mg/ml stock) (Calbiochem, Merck, UK) was applied as a specific blocking peptide when co-incubated 4:1 (v/v) with the anti-nNOS antibody. The 21 amino acid sequence only predicted nNOS by NCBI Blast protein analysis [20]. Alzheimer’s brain tissue was used as a positive control for specific nNOS immunoreactivity [21]. Immunolabelling was visualised by streptavidin biotin-peroxidase labelling with a 3,3′-diaminobenzidine (DAB) chromogen and sections counterstained with Mayer’s haematoxylin.
2.3. Immunofluorescent labelling of nitric oxide synthases in primary epithelial cell culture Otherwise healthy epithelial cells were acquired from nasal brushing biopsies from patients attending a primary ciliary dyskinesia (PCD) diagnostic clinic, and PCD diagnosis had been excluded. Confluent basal epithelial cells were cultured on 12 mm Costar Transwell membranes coated with 300 μg/ml Purecol (Nutacon, The Netherlands) at an air–liquid interface (ALI) for up to 21 days in specialised medium (CC3170, Clonetics, Lonza UK 1:1 with DMEMhigh glucose 4.5 g/l 41966, Invitrogen, UK) with additional 100 nM all-trans retinoic acid (Sigma, UK) until differentiated and ciliated [22]. Transwell membranes containing ALI differentiated ciliated epithelial cells (n = 5 subjects) were excised and fixed in ice cold methanol for 10 minutes. All subsequent steps were carried out at room temperature. Membranes were blocked for 30 minutes in phosphate-buffered saline (PBS) containing 1% bovine serum albumin and 0.05% triton X-100 (blocking buffer) before dissection into segments. Between steps, membrane segments were washed three times in blocking buffer for 5 minutes. All antibodies were diluted with blocking buffer and membrane segments incubated for 90 minutes. The Calbiochem (Merck, UK) primary rabbit polyclonal antibodies verified by published Western Blot: anti-nNOS (1414–1434) [14,23], anti-iNOS (1131–1144) [24,25], anti-eNOS (599–913) [25] (1 mg/ ml stock) were used at 1:200. The synthetic peptide fragment CRSESIAFIEESKKDTDEVFSS corresponding to amino acids 1414– 1434 of nNOS (4 mg/ml stock) (Calbiochem, Merck, UK) was applied as a specific and irrelevant blocking peptide. It was co-incubated 4:1 (v/v) with each anti-NOS antibody. A secondary Alexa488 conjugated goat anti-rabbit antibody (Invitrogen, UK) (2 mg/ml stock) was used at 1:500 in the dark. For co-localisation experiments (n = 3) a mouse monoclonal antiβ-tubulin antibody (ascites fluid) (Sigma, UK), as a marker of cilia [26], was used at 1:500 followed by a secondary Alexa594 conjugated chicken anti-mouse antibody (Invitrogen, UK) (2 mg/ml stock) used at 1:500 in the dark. Membrane segments were finally incubated for 15 minutes in the dark with 5 μM Sytox orange (Invitrogen, UK) or DAPI (Sigma, UK) at 1:500, diluted in PBS, washed and mounted in 100 μl Mowiol between two large glass coverslips to allow easy orientation of membrane segments. Fluorescence was imaged using a laser scanning SP5 confocal microscope (Leica Microsystems, UK).
3. Results 3.1. nNOS localisation in nasal polyp and healthy bronchial tissues by immunohistochemistry In nasal polyp tissue sections (n = 7 subjects) and healthy human bronchial tissue sections (n = 3 subjects) nNOS staining was clearly seen in cilia (Fig. 1b and e). The nNOS immunoreactivity was similar to that of cilia specific β-tubulin staining (Fig. 1g) but was evidently absent from the distal third portion of the cilia (Fig. 1b and e). The nNOS immunoreactivity in the proximal portion of the cilia of both the nasal polyp and healthy bronchial tissue was consistently blocked by the addition of the peptide fragment corresponding to the sequence used to raise the anti-nNOS antibody (Fig. 1c and f). The specificity of the nNOS antibody was verified in paraffin wax embedded Alzheimer’s brain tissue sections, by its appropriate immunolabelling of cell bodies, neurites and blood vessels concentrated at Alzheimer’s plaques (Fig. 1h and i) [21]. Control sections substituting the primary antibody for a primary isotype matched control antibody (Fig. 1a and d) or omitting antibodies (not shown) demonstrated no immunoreactivity. 3.2. nNOS localisation in primary nasal epithelial cell cultures by immunofluorescence labelling Healthy cultures of human primary nasal epithelial cells differentiated and ciliated at ALI and cilia were perpendicular to the epithelial cell layer by confocal microscopy (Fig. 2) and also illustrated by a representative scanning electron microscope image (supplementary Fig. S1a). Neuronal NOS consistently localised to the ciliary compartment of ciliated ALI-cultured epithelial cells (n = 5 subjects) (Fig. 2b) and co-localised with the cilia marker β-tubulin [26], in the proximal part of the ciliary axoneme but not towards the distal portion (Fig. 2c; and supplementary Fig. S1j and l). The immunoreactivities of iNOS and eNOS were demonstrated: diffuse and compartmentalised cytoplasmic iNOS (Fig. 2d–f), and diffuse cytoplasmic eNOS (Fig. 2g–i), with neither localised to the ciliary axoneme as depicted by confocal microscopy orthogonal sectioning (f and i) (n = 5 subjects). The iNOS showed some punctate immunostaining near to the base of the cilia (Fig. 2d–f). The nNOS blocking peptide was co-incubated with the antinNOS antibody and specifically blocked its reactivity (supplementary Fig. S1m–o). The nNOS blocking peptide applied at the same concentration had no blocking or modifying effects on the immunoreactivities of the anti-iNOS antibody (supplementary Fig. S1d–f) or the anti-eNOS antibody (supplementary Fig. S1g–i). Negative controls had counterstained nuclei with primary antibodies omitted and demonstrated no specific immunoreactivity with both secondary antibodies combined (Fig. 2a). Further negative controls included incubating samples with rabbit polyclonal antinNOS antibody with the inappropriate chicken anti-mouse A594 secondary antibody (supplementary Fig. S1b), or mouse monoclonal anti-β-tubulin antibody with the inappropriate goat antirabbit A488 secondary antibody (supplementary Fig. S1c) to show absence of cross-reactivity. 4. Discussion We have demonstrated a novel localisation for nNOS consistently to the proximal portion of cilia in human nasal polyp and healthy bronchial tissue, which co-localised with the cilia marker β-tubulin in healthy bronchial tissue sections. The immunolocalisation of nNOS to cilia was confirmed by its consistent abolition using a blocking peptide, and the antibody specificity was corroborated by demonstrating relevant nNOS
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Fig. 1. nNOS localisation in nasal polyp and healthy bronchial tissues by immunohistochemistry. Immunohistochemical labelling of nNOS and controls in paraffin wax embedded human ciliated nasal polyp tissue (serial sections: a–c), GMA resin embedded healthy human lung tissue (serial sections d–g) and human Alzheimer’s brain tissue (serial sections h and i). Sections were incubated with rabbit polyclonal anti-nNOS antibody followed by a goat anti-rabbit-HRP secondary antibody and DAB substrate and counterstained with haematoxylin. The nNOS immunostaining of cell bodies, neuritis, blood vessels and Alzheimer’s plaques in paraffin wax embedded sections of human Alzheimer’s brain tissue is shown as a control (h and i). Nasal polyp (a) and healthy human lung (d) tissue sections were incubated with an IgG isotype control primary antibody, and cilia on healthy human lung (g) tissue were positively staining using mouse anti-β-tubulin. The nNOS staining was specifically blocked by co-incubation with 4:1 v/v with the anti-nNOS antibody peptide fragment on both tissue types (c and f). (Scale bars = 20 μm).
immunoreactivity in Alzheimer’s brain tissue sections. In control experiments the nNOS blocking peptide did not block the antiiNOS antibody or anti-eNOS antibody immunoreactivities demonstrating blocking specificity for the anti-nNOS antibody alone. Immunofluorescence experiments demonstrated specifically that the nNOS labelling in the proximal portion of cilia did not extend to the tip of the cilia as identified by the β-tubulin immunoreactivity in healthy primary ciliated epithelial cells cultured at ALI. The three NOS isoenzymes nNOS, iNOS and eNOS are present in airway epithelium [4,27] and NO, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) modulate CBF via activation of cAMP and cGMP-dependent kinases [28]. Specifically, eNOS reportedly localises to the basal body of cilia to modulate CBF signalling via cGMP-PKG and cAMP-PKA [28,29] and non-selective NOS inhibition by NG-nitro-L-arginine methyl ester (L-NAME) or NG-monomethyl-l-arginine (L-NMMA) can inhibit CBF stimulation [4,28] but individual NOS inhibition is not reported. As expected [27], iNOS and eNOS isoenzymes were localised within cultured human primary ciliated epithelial cells, but neither localised to the ciliary axoneme. Our data support the contention that nNOS activity in the ciliary axoneme may contribute to CBF modulation,
particularly as nNOS phosphorylation is dependent on PKA [30] also within the axoneme [29]. We speculate that nNOS localisation to the proximal region of the cilia could indicate a role in influencing basal body, transition zone and axoneme structure and function. These sub-ciliary compartments have specific protein compositions that are important for protein entry and exit from the cilium and intraciliary protein trafficking and in particular there are a number of ciliopathies in which protein trafficking is disrupted by mutations in proteins that localise to the transition zone and are required for intraflagella transport (IFT) (reviewed in Reference 31). There is no evidence to suggest that NOS protein mutations are responsible for ciliopathies, but localised nNOS activity and NO production at the proximal portion of the cilium may be relevant to normal protein trafficking. 5. Conclusion We report a novel localisation of nNOS at the proximal portion of cilia in airway epithelium and conclude that its independent and local regulation of NO levels is crucial for normal cilia development and function.
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Fig. 2. nNOS, iNOS and eNOS localisation in primary nasal epithelial cell cultures by immunofluorescence labelling. Primary human airway epithelial cell cultures ciliated at ALI (a–i) were separately immunofluorescently labelled with rabbit polyclonal:anti-nNOS (b and c), anti-iNOS (d–f) and anti-eNOS (g–i) antibodies and an Alexa488 conjugated goat anti-rabbit secondary antibody (green), and nuclei were counterstained with DAPI (blue). Cilia were immunofluorescently labelled with mouse anti-β-tubulin (c, d, f, g and i) antibody and an Alexa594 conjugated chicken anti-mouse secondary antibody (red). As a negative control the primary antibodies were omitted (a). Immunofluorescence labelling was detected using an SP5 laser scanning confocal microscope with a 63× oil immersion objective lens. Images (d, e, g and h) were representative z-plane sections and distribution of immunofluorescence labelling is shown by orthogonal z-sections (f and i). Images (a–c) were maximum projections. (Scale bar = 20 μm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Source of funding This research received funding from Wessex Medical Research M06. The National PCD Centre in Southampton is commissioned and funded by NHS England. Authors’ contributions CLJ designed experiments performed data analysis and wrote the manuscript. JSL and PML contributed to design and manuscript review. WTW, HO and IP performed immunofluorescence and/ or
immunohistochemistry of NOS proteins in airway tissue. All authors read and approved the final manuscript. Acknowledgments The authors are grateful to all volunteers for their participation in this study. The authors are also grateful to Mr Philip Harries and Mr Salil Nair and their surgical teams for their collection of nasal tissues, to Dr Jane Warner for access to healthy bronchial tissue sections and to Dr Delphine Boche for access to Alzheimer’s brain tissue sections. The authors would also like to thank Edith Quinn and staff
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members of the Histochemistry Research Unit, and Dr David Johnston for Confocal Microscopy and Patricia Goggin for the SEM imaging of the Biomedical Imaging Unit. The PCD Centre receives research funding from EU-FP7 funded BESTCILIA 305404; collection of research samples is supported by NIHR Southampton Respiratory Biomedical Research Unit and NIHR Wellcome Trust Clinical Research Facility.
Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.niox.2014.11.003.
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[13] J. Alberty, W. Stoll, C. Rudack, The effect of endogenous nitric oxide on mechanical ciliostimulation of human nasal mucosa, Clin. Exp. Allergy 36 (2006) 1254–1259. [14] D.R. Springall, V. Riveros-Moreno, L. Buttery, A. Suburo, A.E. Bishop, M. Merrett, et al., Immunological detection of nitric oxide synthase(s) in human tissues using heterologous antibodies suggesting different isoforms, Histochemistry 98 (1992) 259–266. [15] K.W. Rosbe, J.W. Mims, J. Prazma, P. Petrusz, A. Rose, A.F. Drake, Immunohistochemical localization of nitric oxide synthase activity in upper respiratory epithelium, Laryngoscope 106 (1996) 1075–1079. [16] B. Degano, S. Valmary, E. Serrano, P. Brousset, J.F. Arnal, Expression of nitric oxide synthases in primary ciliary dyskinesia, Hum. Pathol. 42 (2011) 1855– 1861. [17] F.L. Ricciardolo, M.C. Timmers, P. Geppetti, A. van Schadewijk, J.J. Brahim, J.K. Sont, et al., Allergen-induced impairment of bronchoprotective nitric oxide synthesis in asthma, J. Allergy Clin. Immunol. 108 (2001) 198–204. [18] L. Kobzik, D.S. Bredt, C.J. Lowenstein, J. Drazen, B. Gaston, D. Sugarbaker, et al., Nitric oxide synthase in human and rat lung: immunocytochemical and histochemical localization, Am. J. Respir. Cell Mol. Biol. 9 (1993) 371–377. [19] W. Coers, W. Timens, C. Kempinga, P.A. Klok, H. Moshage, Specificity of antibodies to nitric oxide synthase isoforms in human, guinea pig, rat, and mouse tissues, J. Histochem. Cytochem. 46 (1998) 1385–1392. [20] RID: 1GFU4ZR001N. , 2014 (accessed 16.09.14). [21] V. Thorns, L. Hansen, E. Masliah, nNOS expressing neurons in the entorhinal cortex and hippocampus are affected in patients with Alzheimer’s disease, Exp. Neurol. 150 (1998) 14–20. [22] R.A. Hirst, C.L. Jackson, J.L. Coles, G. Williams, A. Rutman, P.M. Goggin, et al., Culture of primary ciliary dyskinesia epithelial cells at air-liquid interface can alter ciliary phenotype but remains a robust and informative diagnostic aid, PLoS ONE 9 (2014) doi:10.1371/journal.pone.e89675. [23] V. Riveros-Moreno, C. Beddell, S. Moncada, Nitric oxide synthase. Structural studies using anti-peptide antibodies, Eur. J. Biochem. 215 (1993) 801–808. [24] D.S. Bredt, P.M. Hwang, C.E. Glatt, C. Lowenstein, R.R. Reed, S.H. Snyder, Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase, Nature 351 (1991) 714–718. [25] S. Nuedling, R.H. Karas, M.E. Mendelsohn, J.A. Katzenellenbogen, B.S. Katzenellenbogen, R. Meyer, et al., Activation of estrogen receptor β is a prerequisite for estrogen-dependent upregulation of nitric oxide synthases in neonatal rat cardiac myocytes, FEBS Lett. 502 (2001) 103–108. [26] R. Linck, X. Fu, J. Lin, C. Ouch, A. Schefter, P. Warren, et al., Insights into the structure and function of ciliary and flagellar doublet microtubules: tektins, Ca2+-binding proteins, and stable profilaments, J. Biol. Chem. 298 (2014) 17427–17444. [27] K. Asano, C.B. Chee, B. Gaston, C.M. Lily, C. Gerard, J.M. Drazen, et al., Constitutive and inducible nitric oxide synthase gene expression, regulation and activity in human lung epithelial cells, Proc. Natl. Acad. Sci. U.S.A. 91 (1994) 10089– 10093. [28] J.H. Sisson, J.A. Pavlik, T.A. Wyatt, Alcohol stimulates ciliary motility of isolated airway axonemes through a nitric oxide, cyclase, and cyclic nucleotidedependent kinase mechanism, Alcohol. Clin. Exp. Res. 33 (2009) 610–616. [29] S.L. Stout, T.A. Wyatt, J.J. Adams, J.H. Sisson, Nitric oxide-dependent cilia regulatory enzyme localization in bovine bronchial epithelial cells, J. Histochem. Cytochem. 55 (2007) 433–442. [30] J. Yu, L. Yu, Z. Chen, L. Zheng, X. Chen, X. Wang, et al., Protein inhibitor of neuronal nitric oxide synthase interacts with protein kinase A inhibitors, Brain Res. Mol. Brain Res. 2 (2002) 145–149. [31] K. Szymanska, C.A. Johnson, The transition zone: an essential functional compartment of cilia, Cilia 1 (2012) doi:10.1186/2046-2530-1-10.
Contents lists available at ScienceDirect
Nitric Oxide j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y n i o x
Brief Communication
Neuronal NOS localises to human airway cilia Claire L. Jackson a,b,*, Jane S. Lucas a,b, Woolf T. Walker a,b, Holly Owen a, Irnthu Premadeva a, Peter M. Lackie a,b a
Academic Unit of Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, UK Primary Ciliary Dyskinesia Centre, NIHR Southampton Respiratory Biomedical Research Unit, University of Southampton and University Hospital Southampton NHS Foundation Trust, Southampton, UK b
A R T I C L E
I N F O
Article history: Received 25 June 2014 Revised 21 October 2014 Available online 6 November 2014 Keywords: NOS EC 1.14.13.39 nNOS Cilia Airway Epithelium
A B S T R A C T
Background: Airway NO synthase (NOS) isoenzymes are responsible for rapid and localised nitric oxide (NO) production and are expressed in airway epithelium. We sought to determine the localisation of neuronal NOS (nNOS) in airway epithelium due to the paucity of evidence. Methods and results: Sections of healthy human bronchial tissue in glycol methacrylate resin and human nasal polyps in paraffin wax were immunohistochemically labelled and reproducibly demonstrated nNOS immunoreactivity, particularly at the proximal portion of cilia; this immunoreactivity was blocked by a specific nNOS peptide fragment. Healthy human epithelial cells differentiated at an air–liquid interface (ALI) confirmed the presence of all three NOS isoenzymes by immunofluorescence labelling. Only nNOS immunoreactivity was specific to the ciliary axonemeand co-localised with the cilia marker β-tubulin in the proximal part of the ciliary axoneme. Conclusions: We report a novel localisation of nNOS at the proximal portion of cilia in airway epithelium and conclude that its independent and local regulation of NO levels is crucial for normal cilia function. © 2014 Elsevier Inc. All rights reserved.
1. Background Nitric oxide (NO) is a mobile, reactive and ubiquitous signalling molecule within the airway, regulating vascular and bronchial tone, airway permeability [1] and ciliary beat frequency (CBF) [2–5]. It is pro-inflammatory with anti-bacterial action [6,7] and tumouricidal activity [8]. NO is generated from L-arginine by the catalytic activity of nitric oxide synthase (NOS) isoenzymes. The NOS isoenzymes, encoded by NOS1–3 genes on different chromosomes, include neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) respectively. NOS activity is dependent on the presence of cofactors including nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide, and heme [6,9–12].
NADPH reactivity, iNOS and eNOS are localised in the epithelial cell layer of the healthy and allergic human nasal mucosa [5,13–16]. Bronchial epithelium biopsied from atopic asthmatic subjects demonstrated immunoreactivity for nNOS, iNOS and eNOS throughout the columnar and basal cells, with prominent staining for each at the apical surface of columnar cells [17]. Neuronal NOS is also reported in nervous, vascular, gut epithelium but there is little data from nasal or bronchial epithelium, with no reports of localisation in cilia [14,16–19]. The aim of our study was to determine the expression of nNOS protein in healthy human airway ciliated epithelium. 2. Methods 2.1. Ethics statement
Abbreviations: ALI, air–liquid interface; BEGM, bronchial epithelial cell growth medium; CBF, ciliary beat frequency; cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; DAB, 3,3′-diaminobenzidine; GMA, glycol methacrylate; IFT, intraflagella transport; IgG, immunoglobulin G; IL-1β, interleukin-1 beta; NADPH, nicotinamide adenine dinucleotide phosphate; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NOS, nitric oxide synthase; NOS1–3, neuronal nitric oxide synthase 1–3; PBS, phosphate-buffered saline; PKA, protein kinase A; PKIs, protein kinase A inhibitors; PIN, protein inhibitors of neuronal nitric oxide synthase; TBS, tris-buffered saline. * Corresponding author. Clinical and Experimental Sciences, University of Southampton Faculty of Medicine, Southampton General Hospital, Mailpoint 12, Level B South Block, Tremona Road, Southampton SO16 6YD, UK. Fax: 023 8120 5230. E-mail address: [email protected] (C.L. Jackson). http://dx.doi.org/10.1016/j.niox.2014.11.003 1089-8603/© 2014 Elsevier Inc. All rights reserved.
Our research received approval from the National Research Ethics Service (NRES), South Central Committee (06/Q1702/109) and locally at the University Hospital Southampton NHS Foundation Trust Research and Development Department (RHMCH10395). All subjects gave written informed consent. 2.2. Immunohistochemical labelling of nitric oxide synthases Human nasal polyp (n = 7 subjects) in paraffin wax sections and healthy human bronchial tissue (n = 3 subjects) in glycol
4
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methacrylate (GMA) resin sections were immunostained using standard protocols. For paraffin wax sections, antibodies were applied following heat based antigen retrieval in 10 mM citrate buffer (pH 6.0). The Calbiochem (Merck, UK) primary rabbit polyclonal antinNOS (1414–1434) antibody (1 mg/ml stock) was used at 1:1500 for paraffin wax sections and 1:100 for nNOS in GMA resin sections and incubated overnight at 4 °C. A primary mouse monoclonal β-tubulin antibody (Sigma) (ascites fluid) used at 1:15,000 was used as a positive control for cilia staining in GMA resin sections. Equivalent concentrations of an anti-IgG isotype antibody were used as a negative control. The synthetic peptide fragment CRSESIAFIEESKKDTDEVFSS corresponding to amino acids 1414– 1434 of nNOS (4 mg/ml stock) (Calbiochem, Merck, UK) was applied as a specific blocking peptide when co-incubated 4:1 (v/v) with the anti-nNOS antibody. The 21 amino acid sequence only predicted nNOS by NCBI Blast protein analysis [20]. Alzheimer’s brain tissue was used as a positive control for specific nNOS immunoreactivity [21]. Immunolabelling was visualised by streptavidin biotin-peroxidase labelling with a 3,3′-diaminobenzidine (DAB) chromogen and sections counterstained with Mayer’s haematoxylin.
2.3. Immunofluorescent labelling of nitric oxide synthases in primary epithelial cell culture Otherwise healthy epithelial cells were acquired from nasal brushing biopsies from patients attending a primary ciliary dyskinesia (PCD) diagnostic clinic, and PCD diagnosis had been excluded. Confluent basal epithelial cells were cultured on 12 mm Costar Transwell membranes coated with 300 μg/ml Purecol (Nutacon, The Netherlands) at an air–liquid interface (ALI) for up to 21 days in specialised medium (CC3170, Clonetics, Lonza UK 1:1 with DMEMhigh glucose 4.5 g/l 41966, Invitrogen, UK) with additional 100 nM all-trans retinoic acid (Sigma, UK) until differentiated and ciliated [22]. Transwell membranes containing ALI differentiated ciliated epithelial cells (n = 5 subjects) were excised and fixed in ice cold methanol for 10 minutes. All subsequent steps were carried out at room temperature. Membranes were blocked for 30 minutes in phosphate-buffered saline (PBS) containing 1% bovine serum albumin and 0.05% triton X-100 (blocking buffer) before dissection into segments. Between steps, membrane segments were washed three times in blocking buffer for 5 minutes. All antibodies were diluted with blocking buffer and membrane segments incubated for 90 minutes. The Calbiochem (Merck, UK) primary rabbit polyclonal antibodies verified by published Western Blot: anti-nNOS (1414–1434) [14,23], anti-iNOS (1131–1144) [24,25], anti-eNOS (599–913) [25] (1 mg/ ml stock) were used at 1:200. The synthetic peptide fragment CRSESIAFIEESKKDTDEVFSS corresponding to amino acids 1414– 1434 of nNOS (4 mg/ml stock) (Calbiochem, Merck, UK) was applied as a specific and irrelevant blocking peptide. It was co-incubated 4:1 (v/v) with each anti-NOS antibody. A secondary Alexa488 conjugated goat anti-rabbit antibody (Invitrogen, UK) (2 mg/ml stock) was used at 1:500 in the dark. For co-localisation experiments (n = 3) a mouse monoclonal antiβ-tubulin antibody (ascites fluid) (Sigma, UK), as a marker of cilia [26], was used at 1:500 followed by a secondary Alexa594 conjugated chicken anti-mouse antibody (Invitrogen, UK) (2 mg/ml stock) used at 1:500 in the dark. Membrane segments were finally incubated for 15 minutes in the dark with 5 μM Sytox orange (Invitrogen, UK) or DAPI (Sigma, UK) at 1:500, diluted in PBS, washed and mounted in 100 μl Mowiol between two large glass coverslips to allow easy orientation of membrane segments. Fluorescence was imaged using a laser scanning SP5 confocal microscope (Leica Microsystems, UK).
3. Results 3.1. nNOS localisation in nasal polyp and healthy bronchial tissues by immunohistochemistry In nasal polyp tissue sections (n = 7 subjects) and healthy human bronchial tissue sections (n = 3 subjects) nNOS staining was clearly seen in cilia (Fig. 1b and e). The nNOS immunoreactivity was similar to that of cilia specific β-tubulin staining (Fig. 1g) but was evidently absent from the distal third portion of the cilia (Fig. 1b and e). The nNOS immunoreactivity in the proximal portion of the cilia of both the nasal polyp and healthy bronchial tissue was consistently blocked by the addition of the peptide fragment corresponding to the sequence used to raise the anti-nNOS antibody (Fig. 1c and f). The specificity of the nNOS antibody was verified in paraffin wax embedded Alzheimer’s brain tissue sections, by its appropriate immunolabelling of cell bodies, neurites and blood vessels concentrated at Alzheimer’s plaques (Fig. 1h and i) [21]. Control sections substituting the primary antibody for a primary isotype matched control antibody (Fig. 1a and d) or omitting antibodies (not shown) demonstrated no immunoreactivity. 3.2. nNOS localisation in primary nasal epithelial cell cultures by immunofluorescence labelling Healthy cultures of human primary nasal epithelial cells differentiated and ciliated at ALI and cilia were perpendicular to the epithelial cell layer by confocal microscopy (Fig. 2) and also illustrated by a representative scanning electron microscope image (supplementary Fig. S1a). Neuronal NOS consistently localised to the ciliary compartment of ciliated ALI-cultured epithelial cells (n = 5 subjects) (Fig. 2b) and co-localised with the cilia marker β-tubulin [26], in the proximal part of the ciliary axoneme but not towards the distal portion (Fig. 2c; and supplementary Fig. S1j and l). The immunoreactivities of iNOS and eNOS were demonstrated: diffuse and compartmentalised cytoplasmic iNOS (Fig. 2d–f), and diffuse cytoplasmic eNOS (Fig. 2g–i), with neither localised to the ciliary axoneme as depicted by confocal microscopy orthogonal sectioning (f and i) (n = 5 subjects). The iNOS showed some punctate immunostaining near to the base of the cilia (Fig. 2d–f). The nNOS blocking peptide was co-incubated with the antinNOS antibody and specifically blocked its reactivity (supplementary Fig. S1m–o). The nNOS blocking peptide applied at the same concentration had no blocking or modifying effects on the immunoreactivities of the anti-iNOS antibody (supplementary Fig. S1d–f) or the anti-eNOS antibody (supplementary Fig. S1g–i). Negative controls had counterstained nuclei with primary antibodies omitted and demonstrated no specific immunoreactivity with both secondary antibodies combined (Fig. 2a). Further negative controls included incubating samples with rabbit polyclonal antinNOS antibody with the inappropriate chicken anti-mouse A594 secondary antibody (supplementary Fig. S1b), or mouse monoclonal anti-β-tubulin antibody with the inappropriate goat antirabbit A488 secondary antibody (supplementary Fig. S1c) to show absence of cross-reactivity. 4. Discussion We have demonstrated a novel localisation for nNOS consistently to the proximal portion of cilia in human nasal polyp and healthy bronchial tissue, which co-localised with the cilia marker β-tubulin in healthy bronchial tissue sections. The immunolocalisation of nNOS to cilia was confirmed by its consistent abolition using a blocking peptide, and the antibody specificity was corroborated by demonstrating relevant nNOS
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Fig. 1. nNOS localisation in nasal polyp and healthy bronchial tissues by immunohistochemistry. Immunohistochemical labelling of nNOS and controls in paraffin wax embedded human ciliated nasal polyp tissue (serial sections: a–c), GMA resin embedded healthy human lung tissue (serial sections d–g) and human Alzheimer’s brain tissue (serial sections h and i). Sections were incubated with rabbit polyclonal anti-nNOS antibody followed by a goat anti-rabbit-HRP secondary antibody and DAB substrate and counterstained with haematoxylin. The nNOS immunostaining of cell bodies, neuritis, blood vessels and Alzheimer’s plaques in paraffin wax embedded sections of human Alzheimer’s brain tissue is shown as a control (h and i). Nasal polyp (a) and healthy human lung (d) tissue sections were incubated with an IgG isotype control primary antibody, and cilia on healthy human lung (g) tissue were positively staining using mouse anti-β-tubulin. The nNOS staining was specifically blocked by co-incubation with 4:1 v/v with the anti-nNOS antibody peptide fragment on both tissue types (c and f). (Scale bars = 20 μm).
immunoreactivity in Alzheimer’s brain tissue sections. In control experiments the nNOS blocking peptide did not block the antiiNOS antibody or anti-eNOS antibody immunoreactivities demonstrating blocking specificity for the anti-nNOS antibody alone. Immunofluorescence experiments demonstrated specifically that the nNOS labelling in the proximal portion of cilia did not extend to the tip of the cilia as identified by the β-tubulin immunoreactivity in healthy primary ciliated epithelial cells cultured at ALI. The three NOS isoenzymes nNOS, iNOS and eNOS are present in airway epithelium [4,27] and NO, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) modulate CBF via activation of cAMP and cGMP-dependent kinases [28]. Specifically, eNOS reportedly localises to the basal body of cilia to modulate CBF signalling via cGMP-PKG and cAMP-PKA [28,29] and non-selective NOS inhibition by NG-nitro-L-arginine methyl ester (L-NAME) or NG-monomethyl-l-arginine (L-NMMA) can inhibit CBF stimulation [4,28] but individual NOS inhibition is not reported. As expected [27], iNOS and eNOS isoenzymes were localised within cultured human primary ciliated epithelial cells, but neither localised to the ciliary axoneme. Our data support the contention that nNOS activity in the ciliary axoneme may contribute to CBF modulation,
particularly as nNOS phosphorylation is dependent on PKA [30] also within the axoneme [29]. We speculate that nNOS localisation to the proximal region of the cilia could indicate a role in influencing basal body, transition zone and axoneme structure and function. These sub-ciliary compartments have specific protein compositions that are important for protein entry and exit from the cilium and intraciliary protein trafficking and in particular there are a number of ciliopathies in which protein trafficking is disrupted by mutations in proteins that localise to the transition zone and are required for intraflagella transport (IFT) (reviewed in Reference 31). There is no evidence to suggest that NOS protein mutations are responsible for ciliopathies, but localised nNOS activity and NO production at the proximal portion of the cilium may be relevant to normal protein trafficking. 5. Conclusion We report a novel localisation of nNOS at the proximal portion of cilia in airway epithelium and conclude that its independent and local regulation of NO levels is crucial for normal cilia development and function.
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Fig. 2. nNOS, iNOS and eNOS localisation in primary nasal epithelial cell cultures by immunofluorescence labelling. Primary human airway epithelial cell cultures ciliated at ALI (a–i) were separately immunofluorescently labelled with rabbit polyclonal:anti-nNOS (b and c), anti-iNOS (d–f) and anti-eNOS (g–i) antibodies and an Alexa488 conjugated goat anti-rabbit secondary antibody (green), and nuclei were counterstained with DAPI (blue). Cilia were immunofluorescently labelled with mouse anti-β-tubulin (c, d, f, g and i) antibody and an Alexa594 conjugated chicken anti-mouse secondary antibody (red). As a negative control the primary antibodies were omitted (a). Immunofluorescence labelling was detected using an SP5 laser scanning confocal microscope with a 63× oil immersion objective lens. Images (d, e, g and h) were representative z-plane sections and distribution of immunofluorescence labelling is shown by orthogonal z-sections (f and i). Images (a–c) were maximum projections. (Scale bar = 20 μm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Source of funding This research received funding from Wessex Medical Research M06. The National PCD Centre in Southampton is commissioned and funded by NHS England. Authors’ contributions CLJ designed experiments performed data analysis and wrote the manuscript. JSL and PML contributed to design and manuscript review. WTW, HO and IP performed immunofluorescence and/ or
immunohistochemistry of NOS proteins in airway tissue. All authors read and approved the final manuscript. Acknowledgments The authors are grateful to all volunteers for their participation in this study. The authors are also grateful to Mr Philip Harries and Mr Salil Nair and their surgical teams for their collection of nasal tissues, to Dr Jane Warner for access to healthy bronchial tissue sections and to Dr Delphine Boche for access to Alzheimer’s brain tissue sections. The authors would also like to thank Edith Quinn and staff
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members of the Histochemistry Research Unit, and Dr David Johnston for Confocal Microscopy and Patricia Goggin for the SEM imaging of the Biomedical Imaging Unit. The PCD Centre receives research funding from EU-FP7 funded BESTCILIA 305404; collection of research samples is supported by NIHR Southampton Respiratory Biomedical Research Unit and NIHR Wellcome Trust Clinical Research Facility.
Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.niox.2014.11.003.
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