Introduction
The purpose of this article is to evaluate the effect of the mechanical force of a sneeze on sinonasal cilia function and determine the molecular mechanism responsible for eliciting the ciliary response to a sneeze.
A novel model was developed to deliver a stimulation simulating a sneeze at 26°C to the
apical surface of mouse and human nasal epithelial cells. Ciliary beating was visualized, and changes in
ciliary beat frequency (CBF) were determined. To interrogate the molecular cascades driving sneeze-induced changes of CBF, pharmacologic manipulation of intra- and extracellular calcium,purinergic,PKA and nitric oxide (NO) signaling were performed. CBF rapidly increases by >150% in response to a sneeze, which is dependent on the release of adenosine triphosphate (ATP), calcium influx, and PKA activation. Furthermore, apical release of ATP is independent of calcium influx, but calcium influx and subsequent increase in CBF are dependent on the ATP release.
Lastly, we observed a blunted ciliary response in surgical specimens derived from patients with chronic rhinosinusitis compared to control patients. Apical ATP release with subsequent calcium mobilization and PKA activation are involved in sinonasal ciliary response to sneezing, which is blunted in patients with upper-airway disease.

Inhaled pathogens, toxins and particulate matter continuously challenge the respiratory system. The
initial mode of defense against these environmental challenges is mucociliary clearance (MCC), which is
composed of two principle components, mucus production and mucus transport. The production
and secretion of mucus generates a surface overlying the airway epithelium that traps and neutralizes inhaled microbes and particulate debris. Ciliary beating propels debris-laden mucus toward the oropharynx, from which it is cleared by expectoration or swallowing. As is evident by several diseases, such as primary ciliary dyskinesia and cystic fibrosis, impaired MCC predisposes the respiratory system to recurrent and/or chronic infections . While multiple cell types in the respiratory epithelium contribute to the production of mucus, the ciliated epithelial cell is solely responsible for generating the mechanical force driving MCC. Ciliary beating can be divided into spontaneous and enhanced beating after physiological stimulation, both of which are important to MCC. When the MCC apparatus is compromised or overwhelmed by environmental challenges, compensatory mechanisms are recruited to help with mucus clearance. This is accomplished in the lower airways with coughing, and in the upper airways by sneezing or nose blowing. Thus, diseases that affect MCC often present with increased coughing and/or sneezing.Chronic rhinosinusitis and allergic rhinitis are common diseases, with a 14 and 35% prevalence, respectively, that manifest with increased frequency of sneezing. In animal models of allergic rhinitis,induction of frequent sneezing is used to confirm establishment of allergic rhinitis. The functionaleffect of sneezing is to expel static or retained nasal respiratory secretions. During this maneuver, there is a transient increase in pressure on the sinonasal epithelium. Several studies have demonstrated the presence of mechanosensors on respiratory ciliated epithelial
cells, which, when activated, increase the cell’s ciliarybeat frequency, a change that would
be beneficial for secretion clearance. To assess whetherupper respiratory ciliated cells respond to sudden
pressure changes induced by sneezing or nose blowing, a novel experimental model was developed.

In vitro sneeze Culture of mouse nasal septal epithelium at an ALI has been described in prior studies. For this new experiment, human epithelial tissue was used.Selection criterion for recruitment was scheduled sinonasal surgery. Exclusion criteria included a history of systemic granulomatous disease, active smoking, or the diagnosis of primary ciliary dyskinesia. Patients with chronic rhinosinusitis met the objective and subjective guidelines for chronic rhinosinusitis outlined by the clinical practice guideline for adult sinusitis set forth by the Academy of Otolaryngology–Head and Neck Surgery. Control patients had no history of chronic rhinosinsusitis and no evidence of sinonasal mucosal inflammation or infection on nasal endoscopy. All patients recruited for the study had stopped all intranasal medication.

Experimental setup for generation and delivery of the sneeze stimulation to ALI cultures. A) Compressed air controlled by Pico-Spritzer was delivered to the surface of ALI cultures, and CBF was imaged with ×63 objective using differential contrast microscopy. B) Tip of the catheter was 6 mm above ALI and 1 mm from the wall of insert chamber. C) Cilia in the central area (b) were used for CBF collection, and the air delivery catheter was on the diameter (a) of transwell.

Methods

All the experiments were performed at 26°C to ensure a steady baseline frequency during the experiment. To keep the air pressure delivered to the cell surface the same in all experiments, the catheter was connected via a rigid plastic boom to a micromanipulator, allowing for accurate, precise, and reproducible placement 6 mm above the apical fluid and 1 mm from the wall of the transwell, thus leaving the center of the transwell unobstructed for observation and video recording. All experiments used cells in the center of the transwell filter. Images were visualized using a ×63 objective on an inverted microscope (Leica Microsystems, Bannockburn, IL, USA).

For ciliary analysis of human explants, tissue obtained during surgery was washed. Next, it was fixed. This fixation of the tissue prevented movement of the mucosa during and after the application of the sneeze stimulus. Once fixed, the tissue was submerged under 0.5 ml medium, and the catheter was positioned. Then CBF, Adenosine Triphosphate release, intracellular calcium dynamics were measured.

Results
h3. Stimulation of CBF by an air puff (sneeze)
Ten ALI cultures were subjected to air puffs with increasing pressure. Because it was noted a fluctuation in baseline CBF of 5%, we chose an increase in CBF of 15% above the average baseline to define stimulation. Increasing the pressure for the air puff, with a 50-ms duration, demonstrated that CBF can be stimulated in a pressure-dependent manner. We then held the pressure constant at 55 mmHg and varied the duration of the air puff, demonstrating a time-dependent stimulation as well. All 10 cultures responded with a ≥15% increase in CBF with 55 mmHg delivered for 50 ms, and thus, these parameters were used for stimulation for the duration of the experiments. Furthermore, these parameters are consistent with the values measured for intranasal pressure and duration during a human sneeze.

Apical pressure-dependent modulation of CBF. A) Well-differentiated murine septal ALI cultures were subjected to designated apical pressure delivered as a 50-ms puff. B) ALI cultures were subjected to increasing duration of air puff with the pressure fixed as 55 mmHg. Each group involved 10 cultures. A culture was deemed stimulated if the baseline CBF increased by >15%.
ATP-dependent response
Prior work has demonstrated that shear stress applied to the apical surface of respiratory epithelial cells stimulates release of ATP into the airway surface liquid.

ATP-dependent response. A) ATP concentration was increased from 498.20 ± 86.63 to 1502.00 ± 233.50 nM (n=5/group) subsequent to applying a sneeze to the cultures. B) CBF to 192.00 ± 14.00% immediately after adding 100 mM ATP at 125 s (n=3), while adding the vehicle solution (PBS) did not change CBF (102.00 ± 1.00%; n=3). C) Left panel: representative tracing of the effects of preincubation with apyrase (50 U/ml) for 20 min prior to applying the sneeze. Right panel: apyrase blunted the sneeze-induced CBF increase from 180.00 ± 18.32% (vehicle; n=6) to 112.00 ± 4.53% (apyrase; n=6). D) Left panel: representative tracing with and without preincubation with suramin (1 mM) for 30 min, demonstrating inhibition of sneeze-induced CBF increase by suramin. Right panel: increase in CBF in vehicle and suramin groups was 162.30 ± 6.24 and 109.50 ± 3.27% (n=8/group), respectively. Results are means ± se. **P < 0.01.
Calcium-dependent response
ATP binds purinergic receptors at the airway surface to activate calcium-mediated second messenger cascades. To ascertain whether this mechanism was active in our cells, it was used the calcium-sensitive dye Fluo-4 to measure changes in intracellular calcium activity in response to the sneeze stimulus.

Calcium influx after a sneeze. Left panel: images of intracellular calcium before and at several time points after puffing (0.00 s before puffing; 2.71, 5.42, and 8.13 s after puffing). Right panel: intracellular calcium increased to a peak of 303.85 ± 49.20% over the presneeze intracellular calcium (n=3) immediately after puff.
Relationship between ATP and calcium
The above experiments demonstrated that ATP release and intracellular calcium elevation occur in the sinonasal epithelium in response to application of an apical air puff: a sneeze (50 mmHg for 50 ms). We wanted to determine whether the rise in intracellular calcium was driving the ATP release or, vice versa, the ATP was driving the rise in intracellular calcium. Thus, we initially chelated extracellular and intracellular calcium with EGTA and BAPTA-AM, and performed the sneeze stimulation with subsequent collection of the apical surface fluid and quantification of the released ATP. Calcium chelation does not affect the release of ATP following the sneeze stimulation, as no difference was found between the control group and the experiment group.

Blunted stimulation of CBF in chronic rhinosinusitis.
Since many rhinopathologies manifest with increasing frequency of sneezing, it was evaluated sinonasal explants from patients with and without chronic rhinosinusitis. It was previously shown that ciliary alterations associated with chronic rhinosinusitis are reversible when sinonasal tissue is maintained in tissue culture conditions. Thus, we used mucosal explants immediately after acquisition in the operating room. It should be noted that explant tissue does not manifest as a robust response to the air-puff stimulation as the primary cultures grown at an ALI. Sinonasal explants obtained from patients with chronic rhinosinusitis evinced a puff stimulation of only 103 ± 1% , while explants from patients without chronic rhinosinusitis evinced a stimulation of 121 ± 3% with no statistical difference in basal rates between CRS and control.

Discussion
MCC is the primary physical host defense mechanism that protects the respiratory system from inhaled microbes and debris. In the upper airway, a sneeze reflex is stimulated when MCC is compromised or overwhelmed. To the best of our knowledge, the current study is the first to describe an in vitro paradigm to model the effects of sneezing on respiratory epithelium. the results demonstrate that this form of mechanical stimulation directly and robustly activates CBF through a mechanism involving release of apical ATP, an increase in intracellular calcium concentration. Our results reveal important mechanistic insights into the physiological effects of sneezing and demonstrate that sneeze-activated CBF stimulation and resultant increased MCC may be involved in the pathology of human upper airway diseases.
The simulated sneeze parameters that were used in the experiments are consistent with the intranasal pressures measured in adults during sneezing. Prior work has demonstrated that lower airway respiratory epithelium can release ATP under cyclic compressive stress and cellular deformation. Following the sneeze, extracellular ATP increased >3 times, accompanied by an increase in CBF. Furthermore, our results demonstrate that quenching the rise in extracellular ATP by pretreating with the ATPase apyrase blunts the sneeze-induced increase in CBF. This strongly suggests that ATP release is required for the ciliary response to the sneeze stimulus.
The mechanism of CBF regulation by calcium remains controversial; while some studies suggest that calcium can regulate CBF directly, other studies have indicated that calcium always works through kinases or phosphatases. Supporting this latter notion are data from patch-clamp experiments under whole cell configuration, which demonstrated that ciliated cells require the presence of cyclic nucleotides for calcium to effectively elevate CBF.
Sneezing is a reflexive compensatory mechanism to help clear retained or excessive retained respiratory secretions from the upper airway. We demonstrate that in addition to forcibly expelling the mucus within the upper airway, the sneeze also activates the respiratory epithelium to increase MCC. A limitation of the study is that our model does not take into account the complex interplay of the respiratory epithelium, including submucosal glands, thickened mucus, neural input, or the effects of resident inflammatory cells. Nonetheless, our data show that sneezing induces release of ATP onto the apical surface of the airway, generating a rise in intracellular calcium that robustly increases CBF.
This article includes supplemental data.

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Published on August 2012 The FASEB Journal to obtain this information.