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28 Testing ofTransport andMeasurement ofCiliary Activity
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365
spectral structure of its intensity uctuations can
be analyzed to provide quantitative information
regarding the frequency and synchrony of the
ciliary beat [5].
Other techniques are under development for
measuring ciliary activity invivo.
28.3.2 In Vitro
CBF is 8 Hz at room temperature and 12 Hz at
37°C.
In vitro brushings from the nasal cavity or
biopsy samples taken from the inferior border of
the middle turbinate or from the inferior turbinate
can be evaluated for coordinated ciliary beating
and ciliary beat frequency (CBF). Using microscope photometry [6], CBF can be deduced from
fast Fourier transform analysis of the light scattering. Normal CBF values depend on the temperature with normal values around 8Hz at room
temperature and 12Hz at 37°C.
The introduction of high-speed cameras (up to
500Hz) created new possibilities [7, 8]: not only
ciliary beat pattern analysis but also amplitude,
degree, and speed of ciliary beat cycle. Also eld
analysis with measurement of ciliary coordination on a whole area of ciliated cells has become
available [9].
For clinical use only CBF is used, and one
should realize that there is no good correlation
between CBF and mucociliary transport velocity [10].
28.4 Additional Testing
28.4.1 Nasal Nitric Oxide
Nasal nitric oxide (nNO) measurement is a good
screening tool for PCD.
Nasal nitric oxide (nNO) was found to be tenfold lower in PCD patients than in control
patients, and it can be used as an easy screening
test for the diagnosis of PCD.It is a noninvasive
technique, but as it requires cooperation of the
patient (breath holding for stable plateau measurements), it is almost impossible to use it below
5years of age. nNO measurement cannot be used
neither for exclusion of PCD nor for proof of
PCD since normal values can be observed in
PCD and low values can be caused by other factors, e.g., obstruction [11–15].
28.4.2 Genetic Analysis
Making a diagnosis of primary ciliary dyskinesia
(PCD) remains challenging. Molecular diagnosis
involves time-consuming tissue culturing, cilia
beating measurements, and/or electron microscopy of microtubule structures.
Also at the genetic level, a diagnosis of PCD
has been challenging. PCD is an autosomal recessive disease. Mutations in many different genes
can result in PCD, and from a genetic point the
disease is very heterogeneous. Next-generation
sequencing, including the use of whole exome
sequencing or whole genome DNA sequencing,
has further identied genes causing PCD.So far,
mutations in 50 genes (ARMC4, CCDC103,
CCDC114, CCDC151, CCDC39, CCDC40,
CCDC65, CCNO, CFAP298, CFAP300,
CFAP53, DNAAF1, DNAAF2, DNAAF3,
DNAAF4, DNAAF5, DNAH11, DNAH5,
DNAH6, DNAH8, DNAH9, DNAI1, DNAI2,
DNAJB13, DNAL1, DRC1, ENKUR, GAS2L2,
GAS8, HYDIN, LRRC56, LRRC6, MCIDAS,
MNS1, NEK10, NME8, OFD1, PIH1D3, RPGR,
RSPH1, RSPH3, RSPH4A, RSPH9, SPAG1,
SPEF2, STK36, TEKT1, TTC12, TTC25,
ZMYND10) have been reported to cause
PCD. Mutations in these genes account for
approximately 70% of PCD cases; therefore, further gene discovery is still expected [16, 17]. The
ciliary axoneme is composed of over 250 proteins [18], and a mutation in each of their genes
possibly can result in PCD. It is expected that
several of them will be found to be mutated in
PCD patients.
Next-generation sequencing, especially
exome sequencing, has been become the method
of genetic analysis of PCD patients [19–22].
Only in PCD families in which the PCD mutations have been identied, Sanger sequencing is
still mainly used for genetic analysis of other
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Table 28.1 PCD causing genes classied by associated
ultrastructural defect
Ultrastructural
defect in TEM Mutated genes
ODA deciency ARMC4 CCDC103 CCDC114
CCDC151 DNAH5 DNAH9
DNAI1 DNAI2 DNAL1
LRRC56 NME8 TTC25
ODA + IDA
deciency
Normal ciliary
ultrastructure
Central pair
abnormalities and
radial spokes
Ciliary aplasia CCNO MCIDAS
Unclear TEM DNAH6 DNAH8 NEK10 ODF1
CFAP298 CFAP300 DNAAF1
DNAAF2 DNAAF3 DNAAF4
DNAAF5 LRRC6 PIH1D3
SPAG1 ZMYND10
CCDC65 DNAH11 DRC1
GAS8 HYDIN SPEF2 STK36
TEKT1
CCDC39 CCDC40 DNAJB13
RSPH1 RSPH3 RSPH4A
RSPH9
RPGR GAS2L2 TTC12
b
M. Jorissen and M. Jaspers
members in that family, such as determining
whether a relative is a carrier of that mutation,
or not.
Mutant genes causing PCD can be classied
by location of their protein products in the ciliary
axoneme (e.g., outer dynein arm, radial spoke,
central complex, nexin–dynein regulatory
complex; [22]) or by associated ultrastructural
defect (e.g., absent outer dynein arm, absent
outer and inner dynein arms, central pair abnormalities; Table 28.1). However, mutations in
some genes (such as DNAH11) do not result in a
Fig. 28.1 (a) Schematic representation of a spheroid in
suspension culture. (b) SEM picture of spheroid after
6weeks in suspension
detectable ultrastructural defect on transmission
electron microscopy (TEM).
Worldwide, further studies are being performed to identify candidate genes and to detect
disease causing mutations in these genes. Earlier
diagnosis could help prevent evolution into irreversible lung damage with bronchiectasis.
will dedifferentiate and cilia will get lost completely. A sequential monolayer–suspension culture system can then be used to let epithelial cells
redifferentiate [23] into ciliated cells (see Fig.28.1).
These newly formed cilia do not express the
acquired abnormalities, but the inherited abnormalities (PCD) are expressed in the culture system [24]. As the functional abnormalities are
28.4.3 Cell Culture
also clearly present, this culture can be used for
the diagnosis of PCD [25]. PCD with normal
Evaluation of cilia after ciliogenesis in culture
remains the most reliable diagnostic technique for
PCD.
Epithelial cells from biopsies can be cultured
invitro. During the growth phase, epithelial cells
ultrastructure would easily be missed with classical transmission electron microscopy on bioptic material [26]. Ciliogenesis invitro has also
been achieved by placing the cells in an air–liquid interface culture system [27].
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28.5 Conclusion
The mucociliary transport rate can be measured
invivo using either the saccharine and/or color test
or radioisotope transport testing as well as by measuring ciliary activity invitro. Inborn disorders of
the mucociliary transport as in primary ciliary dyskinesia (PCD) result in the absence of mucociliary
transport. Nasal nitric oxide (nNO) was found to
be tenfold lower in PCD patients. None of these
tests are absolutely reliable for the diagnosis of
inherited abnormalities. Sequential monolayer–
suspension cell culture with dedifferentiation and
redifferentiation of the ciliated epithelium
improves the reliability of PCD diagnosis.
References
1. Andersen I, Camner P, Jensen PL, et al. A comparison of nasal and tracheobronchial clearance. Arch
Environ Health. 1974;29:290–3.
2. Andersen I, Proctor DF.Measurement of nasal mucociliary clearance. Eur J Respir Dis Suppl. 1983;64:37–40.
3. De Boeck K, Proesmans M, Mortelmans L, et al.
Mucociliary transport using 99mTc-albumin colloid:
a reliable screening test for primary ciliary dyskinesia. Thorax. 2005;60:414–7.
4. Marthin JK, Mortensen J, Pressler T, Nielsen
KG. Pulmonary radioaerosol mucociliary clearance
in diagnosis of primary ciliary dyskinesia. Chest.
2007;132:966–76.
5. Svartengren K, Wiman L-G, Thyberg P, etal. Laser
light scattering spectroscopy: a new method to measure tracheobronchial mucociliary activity. Thorax.
1989;44:539–47.
6. Jorissen M, De Brouwer J, Bessems A, et al.
Quantication of ciliary beat frequency by computerized microscope photometry—a preliminary study on
suspension cultures of human nasal epithelia showing
spontaneous ciliogenesis invitro. Leitz Sci Tech Inf.
1992;10:88–93.
7. Sanderson MJ, Dirksen ER.A versatile and quantitative computer-assisted photoelectronic technique
used for the analysis of ciliary beat cycles. Cell Motil.
1985;5:267–92.
8. Chilvers MA, O’Callaghan C.Analysis of ciliary beat
pattern and beat frequency using digital high speed
imaging: comparison with the photomultiplier and
photodiode methods. Thorax. 2000;55:314–7.
9. Dimova S, Maes F, Brewster ME, etal. High-speed
digital imaging method for ciliary beat frequency
measurement. J Pharm Pharmacol. 2005;57:521–6.
10. Jorissen M. Correlations among mucociliary transport, ciliary function and ciliary structure. Am J
Rhinol. 1998;12:53–8.
11. Lundberg JO, Weitzberg E, Nordvall SL, et al.
Primarily nasal origin of exhaled nitric oxide and
absence in Kartagener’s syndrome. Eur Respir J.
1994;7:1501–4.
12. Bush A, Cole P, Hariri M, etal. Primary ciliary dyskinesia: diagnosis and standards of care. Eur Respir J.
1998;12:982–8.
13. Karadag B, James AJ, Gultekin E, et al. Nasal and
lower airway level of nitric oxide in children with primary ciliary dyskinesia. Eur Respir J. 1999;13:1402–5.
14. Lefevere L, Willems T, Lindberg S, Jorissen
M. Nasal nitric oxide. Acta Otorhinolaryngol Belg.
2000;54:271–80.
15. Barbato A, Frischer T, Kuehni CE, etal. Primary ciliary dyskinesia: a consensus statement on diagnostic
and treatment approaches in children. Eur Respir J.
2009;34:1264–76.
16. Zariwala MA, Omran H, Ferkol TW. The emerging genetics of primary ciliary dyskinesia. Proc Am
Thorac Soc. 2011;8:430–3.
17. Leigh MW, Horani A, Kinghorn BA, etal. Primary
ciliary dyskinesia (PCD): a genetic disorder of motile
cilia. Transl Sci Rare Dis. 2019;4:51–75.
18. Ostrowski LE, Blackburn K, Radde KM, etal. A proteomic analysis of human cilia: identication of novel
components. Mol Cell Proteomics. 2002;1:451–65.
19. Berg JS, Evans JP, Leigh MW, etal. Next generation
massively parallel sequencing of targeted exomes to
identify genetic mutations in primary ciliary dyskinesia: implications for application to clinical testing.
Genet Med. 2011;13:218–29.
20. Liu L, Luo H.Whole-exome sequencing identied a
novel compound heterozygous mutation of LRRC6in
a Chinese primary ciliary dyskinesia patient. Biomed
Res Int. 2018;2018:1854269.
21. Zhang W, Li D, Wei S, etal. Whole-exome sequencing identies a novel CCDC151 mutation, c.325GT
(p.E109X), in a patient with primary ciliary dyskinesia and situs inversus. J Hum Genet. 2019;64:249–52.
22. Lucas JS, Davis SD, Omran H, etal. Primary ciliary
dyskinesia in the genomics age. Lancet Respir Med.
2020;8:202–16.
23. Jorissen M, Van der Schueren B, Tyberghein J,
etal. Ciliogenesis and coordinated ciliary beating in
human nasal epithelial cells cultured in vitro. Acta
Otorhinolaryngol Belg. 1989;43(1):67–73.
24. Jorissen M, Willems T, Van der Schueren B, et al.
Secondary ciliary dyskinesia is absent after ciliogenesis in culture. Acta Otorhinolaryngol Belg.
2000a;54:333–42.
25. Jorissen M, Willems T, Van der Schueren B, et al.
Ultrastructural expression of primary ciliary dyskinesia after ciliogenesis in culture. Acta Otorhinolaryngol
Belg. 2000b;54:343–56.
26. Jorissen M, Willems T, Van der Schueren B. Ciliary
function analysis for the diagnosis of primary ciliary
dyskinesia: advantages of ciliogenesis in culture. Acta
Otolaryngol. 2000c;120:291–5.
27. Hirst RA, Rutman A, Williams G, etal. Ciliated air–
liquid cultures as an aid to diagnostic testing of primary ciliary dyskinesia. Chest. 2010;138:1441–7.
Nasal Defensive Proteins:
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Distribution andaBiological
Function
HideyukiKawauchi
29
Core Messages
• There are various defensive mechanisms in
upper respiratory tract mucosal linings, such
as mechanical and functional barriers.
• The mechanical barrier of sinonasal mucosa
consists of mucus, motile cilia, and respiratory epithelial cells linked by adhesion complexes that include tight junctions.
• The functional barrier in sinonasal mucosa
is dynamic and more complex, being
equipped with innate and acquired immune
response among resident cells on the epithelia and immunocompetent cells in sinonasal
submucosa.
• Various types of nasal defensive proteins in
human nasal mucosa are responsible for exerting those defensive mechanisms. Those proteins are essential for a defense system against
various invading pathogens, such as bacteria
and viruses, and modulate allergic or infective
chronic inammations as well.
• Those proteins derived from epithelial cells or
recruited inammatory cells are also one of
the important key players in the pathogenesis
of rhinosinusitis and allergic rhinitis at the
H. Kawauchi (*)
Department of Microbiology, Faculty of Medicine,
Shimane University, Izumo City, Japan
e-mail: kawauchi@med.shimane-u.ac.jp
epithelial linings of nasal cavity and paranasal
sinuses.
• Those defensive proteins could be classied
from constitutional and functional aspects,
such as surfactants, mucins, antimicrobial
peptides, and inammatory cell-derived
enzymes.
• Chemokines, cytokines, and various antibodies can be dealt with as defensive proteins, to
provoke cellular interactions against microbial
infections or pathogenesis of sinonasal persistent inammations.
29.1 General Concept ofNasal
Defensive Proteins andIts
Mechanism ofActions
29.1.1 Surfactants
Surfactant proteins are considered to play an
important role in surfactant metabolism and host
defense mechanisms in mucosal linings of respiratory tract. Surfactant proteins (SPs) are sialoglycoproteins and members of the collectin
family. They are now distinguished as SP-A,
SP-B, SP-C, and SP-D [1]. They are hydrophilic
proteins responsible for innate immunity [1, 2],
as SP-A and SP-D bind various pathogens, such
as bacteria, viruses, and fungi, at the initial phase
of defense line of mucosal linings. On the other
hand, SP-B and SP-C are hydrophobic proteins,
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
Ö. Ö. Celebi, T. M. Önerci (eds.), Nasal Physiology and Pathophysiology of Nasal Disorders,
https://doi.org/10.1007/978-3-031-12386-3_29
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H. Kawauchi
contributing to surfactant function and packing
and organizing of phospholipids [3]. SPs are now
elucidated to be expressed in many mucosal sites,
such as gastric and intestinal mucosa [4], joints,
peritoneum, nasal mucosa [5], maxillary sinus
mucosa [6], and middle ear mucosa as well [6],
employing various experimental methods with
immunohistochemistry, Western blotting, and
reverse transcription-polymerase chain reaction
(RT-PCR).
29.1.2 Mucins
29.1.2.1 Classication
Mucins are high molecular weight glycoproteins,
constituting the major component of mucus
secretions in various mucosal surfaces, such as
sinonasal cavity, middle ear, and Eustachian tube
as well. There are secretory and membraneassociated forms of mucin, and they protect the
epithelial surface and trap pathogenic bacteria
and viruses for mucociliary clearance. Secretory
mucins contribute to the viscid mucus of the
respiratory, gastrointestinal, and reproductive
tracts and typically form extremely large oligomers through linkage of their protein monomers
by disulde bonds. These proteins are secreted
from the cell to form the mucous gel, which
becomes an integral part of the mucociliary escalator. In contrast, the membrane-associated
mucins have a hydrophobic membrane-spanning
domain and have not been observed to form
oligomer complexes. The histochemical prole
of cellular glycoprotein in normal condition is
quite different in regard with airway level, stage
of maturation, and species. A majority of surface
secretory cells contain a glycoprotein consisting
of a protein backbone with sugar side chains,
having terminal sialic acid, galactose residues,
and a variable content of sulfate esters.
29.1.2.2 Mucin Genes
With the development of molecular biological
techniques, the complementary deoxyribonucleic acid (cDNA) sequences of mucin genes can
now be obtained and the amino acid sequences
of the mucin peptide core deduced. Until now,
21 mucin genes (MUC1–MUC21) have been
reported, but later on some of them were found
as the same gene as already reported [7, 8].
Among them, there are at least eight human
mucin genes (MUC1–MUC4, MUC5AC,
MUC5B, MUC6, and MUC7) and one mouse
mucin gene (MUC1) conrmed to be present in
respiratory tract mucosae.
29.1.2.3 Mucin Gene Expression
andUpregulation inAnimal
Model
The specic mechanisms by which pathogens or
mucosal irritants induce the mucin gene upregulation are unknown yet. It could be easily considered that they probably act either by increasing
the transcription rate or decreasing the rate of
degradation of mucin messenger ribonucleic acid
(mRNA). Jany observed human MUC2 gene
expression in SO2-exposed rats [9]. They used
rats exposed to 400ppm SO2 for 3h day-1, 5days
a week, for 1–3weeks, and found increased numbers of goblet cells and visible mucinous secretions in the airway lumen. In addition, they
employed the northern blot analysis using the
total ribonucleic acid (RNA) extracted from the
rat lung and hybridized it with human MUC2
cDNA (SMUC41) and demonstrated an upregulation of the MUC2 gene in the SO2-exposed rats.
29.1.2.4 Localization ofMucin Genes
inHuman Airways
A number of experimental approaches have been
performed with regard to localization of mucin
genes in human airways by in situ hybridization.
As regard lung or bronchial mucosal linings, the
localization of MUC2 gene expression was
reported by Audie etal. [10]. In their study, bronchial mucosae were obtained from four patients,
whose lungs had been surgically resected for cancer. A radiolabeled antisense oligonucleotide
probe corresponding to the tandem repeat domain
of MUC2 was applied on the tissue specimens,
and consequently, it was localized to occasional
goblet cells of the surface epithelium and to submucosal gland ducts, but not to their secretory
acini. However, on the other hand, oligonucleotide probes to MUC4, MUC5B, and MUC5AC
strongly labeled the gland acinar cells. These
data indicate that several distinct mucin genes are
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expressed in the mucosa either by the same or
distinct cells of the epithelium and glands. Li
et al. [11] demonstrated in nasal mucosa that
MUC2 mRNA transcripts are present in ciliated
and basal cells of the surface epithelia, serous and
mucous acini of submucosal glands, and occasionally mononuclear inammatory cells.
Voynow etal. precisely compared expression levels of three mucin genes, MUC1, MUC2, and
MUC5/5AC, in the respiratory tract of patients
with cystic brosis, patients with allergic rhinitis,
and normal individuals [12]. Mucin transcript
levels in nasal epithelial cells free from inammation were quantitated by an MUC mRNA slotblot method. Their elegant study revealed that
MUC5/5 AC mRNA was expressed at ve- to
tenfold greater levels than MUC2 or MUC1 for
all subjects and MUC2 mRNA levels were similar among all subject groups. To be generally
considered, in situ hybridization demonstrated
that MUC5AC-positive mucous cells are populated in upper respiratory epithelium, whereas
MUC5B-positive mucous cells are populated in
mucous glands of upper respiratory epithelia. In
contrast, middle ear epithelial cells are negative
for MUC5AC mRNA transcripts, but spotty
MUC5B mRNA transcripts are identied [13,
14]. These data remind us the transitional process
of mucin member from the lower airway to the
middle ear cavity. Preciado et al. found that
MUC5B mucin is predominant in patients with
chronic otitis media [15, 16] and conrmed that
transitional process. However, other mucins,
such as MUC2, may be involved in middle ear
mucus of animal models [17], but their amount is
limited or undetectable in human [13, 14].
Conclusively, the quantitative study of mucins
and its comparison looks notoriously difcult
and should be evaluated at the semiquantitative
level, because of higher level of glycosylation
representing a posttranslational modication.
29.1.3 Antimicrobial Peptides
The body uids and organized tissues naturally
contain a variety of antimicrobial substances that
kill or inhibit the growth of microorganism. The
sources and activities of a variety of host antimicrobial substances are summarized in Table29.1.
Among them, a low-molecular weight antimicrobial peptide, defensin, is introduced herein in
relation to the various receptors contributing to
innate immunity.
29.1.3.1 Defensin
Defensins are a family of evolutionarily related
vertebrate antimicrobial peptides with a characteristic beta-sheet-rich fold and a framework of
six disulde-linked cysteines. Two main defensing subfamilies, α- and β-defensins, differ in the
length of peptide segments between the six cyste-
Table 29.1 Antimicrobial substance of host origin present in body uids and organized tissues
Substance Common sources Chemical composition Activity
Lysozyme Serum, saliva, tears Protein Bacterial cell lysis
Complement Serum Protein–carbohydrate
Basic proteins Serum Proteins or basic peptides Disruption of bacterial plasma
Lactoferrin and
transferrin
Defensin Epithelial cells,
Peroxidase Saliva, tissues, neutrophils Protein Act with peroxide to cause lethal
Fibronectin Serum, mucosal surfaces Glycoprotein Clearance of bacteria
Interferons Virus-infected cells Protein Resistance to virus infections
Interleukins Macrophages,
Body secretions, serum
epithelial cells
neutrophils
lymphocytes
Cell death or lysis of bacteria;
lipoprotein complex
Glycoprotein Inhibit microbial growth by binding
Oligopeptides Cell death or lysis of bacteria
Protein Cause fever; promote activation of
participates in inammation
membrane
(withholding) iron
oxidation of cells
immune system
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H. Kawauchi
ines and the pairing of the cysteines that are connected by disulde bonds. Defensins are abundant
in cells and tissues that are involved in host
defense mechanism of mucosal linings, such as
respiratory and intestinal mucosa. In many species, the highest concentrations of α-defensins
are found in granules, the storage organelles of
leukocytes [18, 19]. β-Defensins are mainly produced by a variety of epithelial cells, such as lung
and middle ear [20, 21], and 30 genes from
DEFB1 to DEFB136 are currently identied as
human β-defensin family. Human β-defensin 1
(DEFB1) is expressed constitutively, whereas
β-defensin 2 (DEFB4A) is induced by bacterial
molecules [22, 23] as well as cytokines [24].
Paneth cells are another site of high α-defensin
concentration and contain defensin-rich secretory granules. Claeys et al. reported that there
was no baseline detection of human β-defensin
2in any sinus mucosal samples, and no upregulation was measured for human β-defensin 2 in
paranasal sinus mucosa in patients with chronic
sinusitis or nasal polyposis compared with control turbinate mucosa [25]. On the other hand, α-
and β-defensins were detected in human nasal
mucosa by Lee et al. [26]. They examined the
expression of defensins in inferior turbinate
mucosa of normal subjects and inferior turbinate
mucosa and nasal polyps of patients with chronic
sinusitis, employing reverse transcriptionpolymerase chain reaction (RT-PCR) and immunohistochemistry. According to their results,
β-defensin 1 mRNA was expressed in all tissue
samples. β-Defensin 2 mRNA was detected in the
turbinate mucosa and nasal polyps of patients
with chronic sinusitis, but not in normal mucosa.
Its expression level was signicantly higher in
nasal polyps than in turbinate mucosa. α-Defensin
5 and 6 mRNAs were not expressed in any tissues, but α-defensins 1, 2, and 3 were detected in
all tissue samples obtained from patients with
chronic sinusitis. These results suggest that
β-defensin 1 may play a constitutive role in nasal
defenses, whereas α-defensins 1, 2, and 3 and
β-defensin 2 may be induced in response to local
infection or inammation.
The average concentration of defensins in
these epithelial cells reaches the 10–100μg/mL
range [27], but the local concentrations might be
higher because of uneven distribution.
Most defensins show antimicrobial activity
against bacteria and fungi, especially when tested
under low ionic strength conditions [28, 29] and
with low concentrations of divalent cations,
plasma proteins, and other interfering substances.
It should be taken into consideration that under
these optimal conditions, antimicrobial activity is
observed at concentrations as low as 1–10μg/mL
(low micromole). Permeabilization of target
membrane is the crucial step in defensin- mediated
antimicrobial activity and cytotoxicity. It is also
reported that defensins act as an immunomodulatory molecules, inducing IL-8in epithelial cells
and modulating complement activation [30, 31]
and neutrophil apoptosis [32, 33].
29.1.3.2 Defensin Synthesis andIts
Regulation
Defensin synthesis and release are regulated by
various signals, such as microbial signals, developmental signals, and cytokines. Human
β-defensin 1 (DEFB1) is expressed constitutively
with low level, whereas β-defensin 2 (DEFB4A)
is highly induced by bacterial molecules [23, 24]
as well as cytokines [25]. Various signaling pathways are identied to orchestrate β-defensin 2
expression, such as toll/IL-1 receptor (TIR)dependent NF-κB activation, TIR-dependent
MAPK signaling, and NOD-2-dependent NF-κB
activation. Wang etal. actually demonstrated that
airway epithelia regulate expression of human
β-defensin 2 through toll-like receptor 2 [34].
Vora etal. also proved that human β-defensin 2
expression is regulated by toll-like receptor signaling in intestinal epithelial cells and that LPS
and peptidoglycan stimulated β-defensin 2 promoter activation in a TLR4- and TLR2-dependent
manner, respectively [35].
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29.2 Brief Introduction ofTollLike Receptors inNasal
Epithelial Cells andSignaling
Pathway
29.2.1 Distribution ofTLRs
inNasopharyngeal Mucosae
andInvolvement ofIL-15
inInammation
We employed northern blot assay and RT-PCR to
see the TLR distribution (Fig. 29.1) in upper
respiratory mucosa and showed that human nasal
epithelial cells constitutively expressed mRNA
for TLR2, 3, and 6, but not for TLR4 and TLR9
(Fig. 29.2). Lipoprotein used as a pathogenassociated molecular pattern (PAMP) also
induced IL-15 production of respiratory epithelial cells, which strictly depend on TLR2
(Fig.29.3). In Fig.29.3, in northern blot analysis,
IL-15 mRNA was strongly expressed after lipoprotein stimulation. But in contrast, it was not
found after lipid A stimulation as a ligand of
TLR4. IL-15 concentration in the supernatants of
CCL185 was also upregulated after lipoprotein
stimulation in a dose-dependent manner.
Figure29.4 shows NF-κB activation of respiratory epithelial cells in response to lipoprotein. In
Western blot analysis, phosphorylation of IkBalpha is detected in epithelial cells 15 and 30min
after lipoprotein stimulation. Luciferase assay
and DNA-binding assay reveal that NF-κB activity actually correlated with the lipoprotein
concentration.
Fig. 29.1 Toll family proteins
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Fig. 29.2 Expression of TLRs on macrophages and nasal epithelial cells. (a) Northern blot assay. (b) RT-PCR
H. Kawauchi
Fig. 29.3 Lipoprotein inducing IL-15 from respiratory epithelial cells
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Fig. 29.4 NF-kB activation of respiratory epithelial cells in response to lipoprotein and lipid A
375
29.3 Conclusion
In this chapter, I have extensively introduced the signicance of the so-called defensive proteins on the
upper respiratory tract mucosal linings from various
aspects. As you can see, there are so many complex
factors involved in the defense line of our sinonasal
mucosa in accordance with microbial invasion and
pathogenesis of sinonasal disorders through intracellular signaling pathways. Further investigation of
those mechanisms should be absolutely needed for
elucidating pathologies of sinonasal disorders and
seeking for promising therapeutic strategies. To further end, I have intensively described antiviral
immunity with interferons as an appendix, for your
understanding of SARS-CoV-2 virus infection.
Appendix: Antiviral Proteins
andImmunity
fortheUnderstanding ofSARSCoV- 2 Virus Infection
Introduction
We all know viruses are the intracellular parasites, taking place infection at various mucosal
linings or nerve tissue, including central ner-
vous system, by invading to resident cells of the
mammalian body. Viral life cycle consists of a
short period of extracellular phase, and a longer
intracellular period as long as viruses keep replication. Interferon, defensin, complement, and
antiviral antibodies are, respectively, important
as antiviral proteins. And as antiviral cellular
responses, natural killer (NK) cells, cytotoxic T
lymphocytes (CTLs) are elucidated to, respectively, exert non-specic and specic immune
responses [36]. Those immune system composes of three different stages: innate responses
(complement, interferon, NK cells) appear with
a few days after viral infection and suppress
viral replication. Antigen-specic CTL
responses develop a few days later, having peak
response between days 7 and 10 and resolve ongoing intracellular infection. Finally, antiviral
antibodies in serum reach maximum titers and
continue for months to years, and specic those
antibodies provide a protection against reinfection. The concept of “innate immunity” includes
all sorts of defense mechanism that expel infections with little specicity and less clonal
expansion. It works without much adaptation
and no generation of a long- lasting memory.
So, the mammalian innate immune defenses
comprise defensins, the complement system,
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