Cilia are complex and powerful cellular structures that serve a multitude of functions across many types of organisms. In humans, one of the most critical roles of cilia is defense of the airway. The respiratory epithelium is lined with cilia that normally carry out an integrated and coordinated mechanism called mucociliary clearance. Mucociliary clearance, the process by which cilia transport the viscous mucus blanket of the upper airway to the gastrointestinal tract, is the primary means by which the upper airway clears itself of pathogens, allergens, debris, and toxins. The complex structure and regulatory mechanisms that dictate the form and function of normal cilia are not entirely understood, but it is clear that ciliary dysfunction results in impaired respiratory defense. Ciliary dysfunction may be primary, the result of genetic mutations resulting in abnormal cilia structure, or secondary, the result of environmental, infectious or inflammatory stimuli that disrupt normal motility or coordination.
Cilia are a ubiquitous organelle found on diverse cell types including sperm cells of vertebrates and some invertebrates, unicellular protozoa, and several vertebrate epithelial cell types. In mammals, for example, motile cilia found on cells lining the brain ventricles circulate cerebrospinal fluid; cilia in the respiratory tract sweep debris from the upper airway and the lungs; and oviduct cilia move the fertilized egg to the uterus. In addition, epithelial cilia present early in development are involved in left-right axis determination. Some epithelial cells, such as retinal photoreceptor cells and certain renal epithelial cells, possess immotile cilia that are now known to play important sensory roles in cell function. Individuals with motility-impaired cilia or defects in ciliary assembly may have any number of serious disorders, including respiratory disorders, hydrocephaly, retinal degeneration, polycystic kidney disease, liver disease, and infertility.
Sinonasal epithelium
The unique structure of the sinonasal epithelium facilitates normal cilia function and mucociliary clearance, thereby protecting the airway from debris, pathogens, and inhaled toxins. The anterior margin of the nasal vestibule is protected by a stratified squamous epithelium whose protective barrier includes sebaceous glands, sweat glands, and vibrissae. Near the nasal valves there is a histologic transition to pseudostratified columnar ciliated epithelium. Most of the nasal cavity epithelium consists of pseudostratified columnar ciliated cells, whereas the paranasal sinus epithelium is predominantly simple columnar ciliated cells. In addition to the cilia projecting from their apical surface, the epithelial cells are lined with hundreds of immotile microvilli, hairlike projections of actin filaments, 1 to 2 μm in length, that lie beneath the cell membrane. By increasing the total mucosal surface area, the microvilli aid in sinonasal mucus production, sensation, secretion, and warming and humidifying inspired air. Ciliated columnar epithelial cells comprise approximately 80% of sinonasal mucosa, goblet cells that produce mucus approximately 20%, and progenitor basal cells less than 5%.
Mucus
Mucus production and structure are integrally associated with normal cilia function. Just as abnormal mucus production can impair normal cilia function, abnormal cilia function can result in stagnant mucus containing abundant pathogens and debris and result in chronic inflammation. The mucus layer functions to trap inspired pathogens, particulate matter, and cellular debris, and through the process of mucociliary clearance this layer is continuously cleared and reproduced. The superficial layer of mucus is a viscous gel phase that rides along the tips of fully extended cilia. The deep layer is the sol phase that surrounds and bathes the shafts of cilia. The sol phase is a solution of water and electrolytes (Na1, K1, Ca 2+ , Cl − ) of lower viscosity than the gel phase. Mucus is a complex immunologically active substance made of carbohydrates, enzymes, proteins, immunoglobulins, and other active molecules.
The composition of mucus is essential to normal mucociliary clearance, and disorders of mucus production can be debilitating by causing severe secondary ciliary dysfunction. Cystic fibrosis, an autosomal recessive disease resulting from a mutation in a single gene, involves several organ systems and is characterized by defective electrolyte transport resulting in abnormal mucus production. The genetic defect is found in the cystic fibrosis transmembrane conductance regulator gene product, a cyclic adenosine monophosphate–mediated membrane glycoprotein that forms a chloride channel and regulates the open probability of the sodium channel, ENaC. The defective sodium chloride transport yields abnormally viscous mucus. The goblet cells in such patients are subsequently engorged and distended. These patients have severely impaired mucociliary clearance and frequently develop severe recurrent sinopulmonary infections.
Mucus
Mucus production and structure are integrally associated with normal cilia function. Just as abnormal mucus production can impair normal cilia function, abnormal cilia function can result in stagnant mucus containing abundant pathogens and debris and result in chronic inflammation. The mucus layer functions to trap inspired pathogens, particulate matter, and cellular debris, and through the process of mucociliary clearance this layer is continuously cleared and reproduced. The superficial layer of mucus is a viscous gel phase that rides along the tips of fully extended cilia. The deep layer is the sol phase that surrounds and bathes the shafts of cilia. The sol phase is a solution of water and electrolytes (Na1, K1, Ca 2+ , Cl − ) of lower viscosity than the gel phase. Mucus is a complex immunologically active substance made of carbohydrates, enzymes, proteins, immunoglobulins, and other active molecules.
The composition of mucus is essential to normal mucociliary clearance, and disorders of mucus production can be debilitating by causing severe secondary ciliary dysfunction. Cystic fibrosis, an autosomal recessive disease resulting from a mutation in a single gene, involves several organ systems and is characterized by defective electrolyte transport resulting in abnormal mucus production. The genetic defect is found in the cystic fibrosis transmembrane conductance regulator gene product, a cyclic adenosine monophosphate–mediated membrane glycoprotein that forms a chloride channel and regulates the open probability of the sodium channel, ENaC. The defective sodium chloride transport yields abnormally viscous mucus. The goblet cells in such patients are subsequently engorged and distended. These patients have severely impaired mucociliary clearance and frequently develop severe recurrent sinopulmonary infections.
Cilia structure and function
Sinonasal cilia beat in a coordinated manner to clear the paranasal sinus cavities and upper airway of the mucus blanket containing pathogens and debris. Normal cilia are cylindrical projections from the apical surface of epithelial cells, anchored by intracellular basal bodies. Each epithelial cell is lined with approximately 50 to 200 cilia, measuring 5 to 7 μm in length and 0.2 to 0.3 μm in diameter. The cilium is composed of interconnected microtubules bundled into axonemes, and its overlying membrane is continuous with the cell’s plasma membrane. The microtubules are made of protofilaments, which in turn are composed of α- and β-tubulin dimers.
The axonemes of motile cilia contain 2 central singlet microtubules surrounded by 9 doublet microtubules. Each doublet consists of 1 α-tubule, a complete circle of 13 protofilaments, and 1 β-tubule, an incomplete circle of 10 protofilaments. This structure is consistent among the motile cilia of the respiratory epithelium, oviduct, and cerebral ventricular ependymal cells. The 2 central microtubules are attached by paired bridges whereas the peripheral doublets attach to the central pair via radial spoke heads. Each outer doublet interacts with the adjacent outer doublets via inner dynein arms (IDAs), outer dynein arms (ODAs), and nexin, each having a distinct role in the dynamic motion of cilia bending. Activation of the dynein arms generates a sliding motion of 1 microtubule doublet against the adjacent doublet. It is thought that phosphorylation of the ODAs regulates cilia beat frequency while phosphorylation of the IDAs regulates the waveform pattern of beating. Although the function of the radial spoke heads is not entirely understood, it seems they are involved in regionally limiting the sliding between the microtubules during the ciliary stroke, thus converting the sliding motion generated by the dynein arms into a bending motion of the axoneme.
Each cilium has a forward power stroke followed by a recovery stroke. During the power stroke the cilium is fully extended, and the distal tip reaches the viscous outer gel phase of the mucus layer, transmitting directional force to the overlying mucus layer. During the recovery stroke, the cilium bends 90° and sweeps back to its starting point within the thinner periciliary sol phase. The mechanism of ciliary motion depends on a series of adenosine triphosphate (ATP)-dependent molecular motors that cause the outer doublets of the axoneme to slide relative to each other, producing a vectorial force. The coordination of cilia beating is thought to be secondary either to an intracellular calcium wave, via gap junctions between epithelial cells, that drives microtubule interactions, or to a hydrodynamic wave that forces a timed coordination of nearby cilia. Although the mechanism of coordination that results in this metachronous wave is not entirely understood, it is clear that disease states alter the normal function of cilia, thereby disrupting the critical process of mucociliary clearance.
Dynamic regulation
Ciliary activity accelerates in response to various mechanical, chemical, hormonal, pH, and thermal stimuli. Extracellular nucleotides (adenosine and uridine) are especially potent regulators of epithelial functions stimulating mucociliary clearance through mucus secretion, increasing ciliary beat frequency (CBF), and gating ion channels involved in the maintenance of epithelial surface liquid volume. Nucleotides released by the epithelium in response to mechanical and osmotic stimuli work in a paracrine fashion through metabotropic and ionotropic receptors to potentiate mucociliary clearance by recruiting adjacent cells to increase CBF. Furthermore, adrenergic, cholinergic, and peptidergic stimulations have also been demonstrated to stimulate ciliary motility. These environmental and host stimuli are transmitted via surface receptors and channels to trigger activation of second messenger cascades that regulate phosphorylation status of ciliary proteins, thereby modulating the kinetics of microtubules’ sliding relative to each other. Inositol triphosphate (IP 3 ) mediated calcium transients have been correlated with increased CBF. In addition, protein kinase A (PKA), protein kinase G, and a nitric oxide (NO)–dependent mechanism of CBF stimulation have been proposed, while activation of protein kinase C (PKC) appears to decrease CBF. To maintain rapid control of ciliary activity, kinase anchoring proteins, kinases, and phosphatases have been demonstrated to be tightly associated with the axoneme. Experiments using fluorescence resonance energy transfer in primary ciliated cell culture demonstrated direct evidence that activation of PKA coincides with an increase in CBF, and that the return to baseline frequency lags PKA inactivation, indicating that dephosphorylation by phosphatases is required to terminate CBF stimulation.
As mentioned earlier, environmental stimuli also modulate CBF. Small changes in extracellular and intracellular pH can have a profound impact on CBF. An increase in intracellular pH produces an increase in CBF, whereas a decrease in pH produces a decrease in CBF. However, it is not known whether this effect is due to modulation of kinase activity, even though an acidic pH has been demonstrated to inhibit PKA function, or due to direct regulation of the outer dynein arm of the axoneme. Cilia beat has additionally been shown to be influenced by changes in temperature. Multiple investigations have demonstrated a direct correlation between temperature and CBF. Furthermore, the temperature response appears to be mediated through kinase activity as activation of PKC shifts the temperature curve to the left. Lastly, CBF is also regulated by mechanical factors. Direct mechanical stimulation of the cilia promotes an increase in CBF that coincides with an increase in intracellular Ca 2+ . In addition, shear stress applied to the apical surface of mouse tracheal explants stimulates CBF. The characteristics of this observation include a time-dependent component as well as a directional component with stimulation resulting from caudal applied shear stress. Furthermore, the response depends on purinergic receptor activation as well as intracellular Ca 2+ and ATP. Thus, these experiments suggest that CBF in the trachea coincides with the respiratory cycle, that is, CBF increases with inspiration and returns to baseline during exhalation, thereby preventing microaspirations.
Mucociliary clearance
In the paranasal sinuses the coordinated function of cilia propels the mucus layer from the sinuses to the nasal cavity and then to the nasopharynx, where it is subsequently ingested into the gastrointestinal tract. In the maxillary sinus, mucus flows toward the natural sinus ostium in the superior medial wall of the sinus and then drains into the ethmoidal infundibulum. The anterior ethmoid cells drain into the middle meatus, and the posterior ethmoid cells into the superior meatus. The sphenoid sinus drains into the sphenoethmoidal recess. The frontal sinus drains into the infundibulum. The mucociliary flow from the anterior sinuses converges at the osteomeatal complex before traveling posterior to the nasopharynx. Once the mucus layer is in the nasopharynx, further mucociliary action and swallowing assist its ingestion. Microscopic cilia dysfunction at any step of this intricate transport system can result in significant and clinically evident sinonasal pathology.
Cilia dysfunction
Genetic
Normal and effective mucociliary clearance is a critical component of sinonasal immunity and defense that is dependent on proper cilia function. In addition, unlike the lower airways where a compensation for decreased cilia function can be accomplished by a cough, the paranasal sinuses are solely dependent on ciliary function to propel mucus. There are several pathologic states, both genetic and acquired, in which cilia dysfunction results in impaired mucociliary clearance. These conditions are often a result of uncoordinated and dyssynchronous ciliary function or reduced motility of cilia, rather than complete cilia immotility. The hallmarks of cilia dysfunction in the otorhinolaryngologic evaluation are recurrent otitis media, sinus inflammation, and sinopulmonary infections caused by the accumulation of debris and infectious pathogens.
Primary ciliary dyskinesia (PCD) is an inherited disorder of abnormal cilia function that results in dysfunctional ciliary motility and impaired mucociliary clearance, with reported incidence ranging from 1 in 15,000 to 1 in 60,000 live births. Patients with PCD typically present with chronic recurrent infections of the upper and lower airways, sinusitis, and otitis media. PCD and other syndromes of cilia dysfunction often manifest in several organ systems because cilia serve various systemic functions independent of mucociliary clearance. Approximately half of patients with PCD have situs inversus because normal embryonic nodal cilia are integral in the left-right orientation of visceral development. Kartagener syndrome, described in 1933 before the discovery of the true structure and function of cilia, is the clinical triad of chronic sinusitis, bronchiectasis, and situs inversus. In addition, as sperm motility and fallopian tube transport of ova are dependent on normal cilia motility, PCD patients may also suffer from infertility.
On clinical evaluation, PCD patients may present with symptoms and signs of chronic sinusitis, including nasal congestion, sinus pain and tenderness, mucopurulent nasal discharge, and anosmia. These patients often have a history of acute and chronic otitis media, with or without associated hearing loss and tympanic membrane inflammation, perforation, or scarring. PCD patients may have a chronic productive cough with associated bronchospasm, bronchiectasis, and a history of recurrent pneumonia, with associated wheezing, rales, or rhonchi. Physical examination may also reveal dextrocardia secondary to situs inversus. Because of the unusual constellation of clinical features crossing various organ systems, the diagnosis is often delayed even though the first signs may present in infancy. The differential diagnosis includes neonatal respiratory distress, asthma and allergic rhinitis or sinusitis, cystic fibrosis, or primary immunodeficiencies. A diagnosis of PCD can be confirmed by electron microscopy, genetics testing, immunofluorescent analysis, nasal NO measurement, and high-speed videomicroscopy.
PCD is a genetically heterogeneous disorder. It is most commonly autosomal recessive, but other inheritance patterns have been identified. In more than 80% of PCD patients, the primary ciliary defect is in the outer dynein arm, the inner dynein arm, or both. When the outer dynein arm is defective, most but not all cilia are immotile, even if the inner arm is functional. When the inner dynein arm is defective, the cilia beat with an abnormal pattern and reduced amplitude of stroke. Some PCD patients have an abnormality of the central radial spoke complex connecting the 2 central singlet microtubules to the surrounding 9 doublet microtubules. Such cilia have a stiff repetitive beating motion but without sufficient bending to propel the overlying mucus layer. The genetic mutations responsible for PCD have not been completely identified. The DNAI1 , DNAI2 , and DNAH5 genes, for example, are all associated with outer dynein arm defects and have been implicated in the clinical manifestations of PCD, but many outer dynein arm defects have been described in the absence of identified mutations of these genes.
Acquired
Whereas intrinsic factors can alter cilia function with catastrophic sequelae, extrinsic factors such as pollutants and microbial invaders can directly and indirectly, through induction of inflammatory mediators, affect normal cilia function. This is evident in patients with chronic rhinosinusitis (CRS) who experience relentless cycles of infection and inflammation, with clear clinical sequelae of mucociliary dysfunction ( Fig. 1 ).
