Angiotensin II stimulates airway ciliary motility in rabbit cultured tracheal epithelium

We studied the effect of angiotensin II on ciliary activity in cultured rabbit tracheal epithelium in vitro. Administration of angiotensin II (10(-6) M) elicited an increase in ciliary beat frequency (CBF), as assessed by a photoelectric method, from the baseline value of 906 +/- 21 to 1260 +/- 33 beats min-1 (mean +/- SE, P less than 0.001). This ciliostimulatory effect was dose-dependent, with the maximal increase and EC50 value being 35.6 +/- 5.2% (P less than 0.001) and 5 x 10(-12) M respectively. Nifedipine, Ca2(+)-free medium, indomethacin and the phospholipase A2 inhibitor mepacrine, but not nordihydroguaiaretic acid, reduced the change in CBF. The ciliostimulation induced by angiotensin II was abolished by pretreatment of tissues with [Sar1-Ile8]angiotensin II, an angiotensin II receptor antagonist. Angiotensin II did not increase cyclic AMP levels in epithelial cells. These results suggest that angiotensin II interacts with its specific receptors and stimulates airway ciliary activity through a Ca2(+)-dependent prostaglandin release, without affecting intracellular cyclic AMP levels. Thus, angiotensin II may modulate mucociliary transport function in the respiratory tract.     

Stimulation of airway ciliary motility by immunologically activated canine pulmonary macrophages: role of leukotrienes

To elucidate a possible interaction between alveolar macrophages and airway epithelial cells in allergic conditions, we studied the effect of immunologically stimulated macrophages on ciliary beat frequency (CBF) of cultured canine tracheal epithelium by a photoelectric method. Administration of supernatants from macrophages incubated with anti-dinitrophenyl (DNP) IgE antibody and anti-dinitrophenyl-human serum albumin dose-dependently increased ciliary beat frequency, the maximal increase from the baseline being 30.4 +/- 5.0% (mean +/- SE, P less than 0.01), an effect that was accompanied by the release of leukotriene (LT) C4 and leukotriene D4. This ciliostimulation was not affected by pretreatment of macrophages with indomethacin but was inhibited by that with nordihydroguaiaretic acid. Addition of FPL 55712 abolished the response of ciliary beat frequency to the stimulated macrophages, and exogenously administered leukotriene C4 and leukotriene D4 dose-dependently increased ciliary beat frequency. These results suggest that macrophages increase respiratory ciliary motility through the IgE-mediated release of leukotrienes and may modulate mucociliary transport function in the airway.     

Mechanism of hydrogen peroxide-induced inhibition of sheep airway cilia.

To study the effect of the inflammatory mediator hydrogen peroxide (H2O2) on airway ciliary activity, we measured ciliary beat frequency (CBF) in cultured tracheal explants from sheep. Addition of H2O2 (10(-8) to 10(-4) M) produced a concentration-dependent mean (+/- SEM) decrease in CBF between 11.1 +/- 0.4% (P less than 0.01) and 100 +/- 0% (P less than 0.001); at each concentration, the maximal effect was reached by 20 to 25 min. Between 10(-8) and 10(-6) M H2O2, the decrease in CBF was reversible, lactate dehydrogenase (LDH) release was not significantly increased, and major morphologic lesions were not seen. At higher concentrations of H2O2, incomplete recovery of CBF (10(-5) M) or irreversible ciliostasis (10(-4) M) developed, and a significant increase in LDH and morphologic lesions were present. Catalase (2,000 U/ml) and H-7 (10(-5) M), a protein kinase inhibitor, abolished cilioinhibition produced by H2O2 at 10(-6) M and lower concentrations but not at 10(-5) M and higher concentrations. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, caused a dose-dependent (10(-11) to 10(-5) M), reversible decrease in CBF; this effect was abolished by H-7. We suggest that at nonlethal concentrations, H2O2 inhibits the beat frequency of airway epithelial cilia reversibly, through the activation of second messengers, including protein kinase C. This mechanism might contribute to the previously demonstrated impairment of mucociliary clearance in airway inflammation.     

SRS-A leukotrienes decrease the activity of human respiratory cilia

We have studied the effects of the slow reacting substance of anaphylaxis (SRS-A) constituents leukotrienes (LT) C₄ and D₄ on the ciliary activity of human respiratory cells. The ciliary beat frequency on human nasal cells harvested by cell scraping from the inferior turbinate was measured in a blind design by a microphoto-oscillographic technique. A total of 740 ciliated cells from seventy-four cell scrapings were studied. Mean baseline of ciliary beat frequency was 10.2 Hz. The ciliary beat frequency exhibited a pronounced variability in the spontaneous changes between the cell scrapings, yet less so within cell samples from the cell scrapings. We, therefore, evaluated the effect of the test solutions relative to the spontaneous decrease found during simultaneous perfusion with control solution of samples from the same cell scrapings. LTC₄, 3–300 nmol/l, caused a highly significantly dose-related decrease in the ciliary beat frequency by up to approximately 20% as compared to the corresponding control solution. The effect of LTC₄ was significantly inhibited by the SRS-A receptor antagonist FPL 55712 (10μmol/l), but not by indomethacin (10 μmol/l). LTD₄, 300 nmol/l, also decreased the ciliary beat frequency. LTB₄, which is a leukotriene, although without the sulphidopeptide side chain of the SRS-A leukotrienes, did not affect the ciliary beat frequency in a concentration of 100 nmol/l. This would seem to confirm the structure specificity of the elucidated effect of the SRS-A leukotrienes. Histamine (100 μmol/l) did not affect the ciliary beat frequency. The present study demonstrates that the SRS-A leukotrienes have a specific cilio-depressive effect, which may contribute to mucus obstruction in the lower airways in asthma and other chronic airway diseases.     

Endothelin stimulates ciliary beat frequency and chloride secretion in canine cultured tracheal epithelium

Endothelin, an endothelium-derived vasoconstrictor peptide, has recently been reported to exist in airway epithelial cells. To elucidate a possible role of endothelin on epithelial functions, we studied the effects of this peptide on ciliary beat frequency (CBF) and electrical properties of canine cultured tracheal epithelium by a microphoto-oscillation method and Ussing's short-circuited technique, respectively. Endothelin dose-dependently increased CBF, the maximal increase above the baseline value and EC50 being 32.3 +/- 4.0% (P less than 0.001) and 3 nM, respectively. This effect was moderately attenuated by pretreatment of cells with indomethacin and greatly reduced by Ca2(+)-free medium. Addition of endothelin (10(-6) M) to the mucosal bath of Ussing chamber increased short-circuit current from 5.1 +/- 1.2 to 9.4 +/- 1.7 microA/cm2 (P less than 0.05) and potential difference from 2.9 +/- 0.4 to 5.0 +/- 0.9 mV (P less than 0.05), an effect that was inhibited by indomethacin or Ca2(+)-deficiency and was abolished by the Cl transport inhibitor bumetanide or substitution of Cl with iodide in the medium. These results indicate that endothelin stimulates ciliary motility and Cl secretion probably through an increase in cytosolic Ca2+ and partially a prostaglandin synthesis in canine tracheal epithelium, and suggest that this peptide might play a role in modulating airway mucociliary transport functions.     

The inhibitory effects of platelet activating factor (PAF) on ciliary activity of human paranasal sinus mucosa]

The effect of platelet activating factor (PAF) on human paranasal ciliated cells was investigated in vitro. Normal human paranasal sinus mucosa was obtained by surgical procedure and incubated with Eagle's MEM containing 10% FCS in the form of tissue culture. Ciliary activity was viewed at 37 degrees C under an inverted microscope equipped with a thermoregulator and a humidified CO2 chamber, recorded on video tapes and photoelectrically measured. Ciliary inhibition was observed by the treatment with PAF, in a dose dependent manner, at concentrations from 10(-10) M to 10(-6) M. The inhibitory effect of 10(-8) M PAF on ciliary activity was completely blocked when the mucosa was treated with 10(-6) M CV-3988 or 10(-6) M CV-6209 (specific PAF receptor antagonists). By the radioimmunoassay, the concentration of PAF in tissue culture was reduced by half within 12.5 min, and within 60 min it was only 5% of the initial concentration. There was no significant difference in ciliary inhibition between irrigation after a 60 min incubation with 10(-8) M PAF and non-irrigation. These results indicate that PAF inhibited ciliary activity directly and specifically, and induced irreversible damage primarily within the first 60 min after the challenge.     

The in Vitro and in Vivo Effect of a New Non-Halogenated Corticosteroid - Budesonide - Aerosol on Human Ciliary Epithelial Function

The in vitro and in vivo effect on the ciliary epithelial function of a new corticosteroid (budesonide), with a high topical and negligible systemic activity, was investigated. Ciliary function is an important factor in the nasal clearance mechanism. It must not be hampered by drugs or additives. Ciliary beat frequency (CBF) was measured in vitro with a photo-electric method. CBF appeared to be only slightly decreased by the budesonide and placebo aerosols. As there is no significant difference in ciliotoxicity between the aerosols, the small decrease may be caused by the solubilizing excipient. The influence of the aerosols in vivo was measured in human volunteers as changes in mucus transport time (MTT) with the indigocarmine/saccharine sodium method. Ciliotoxicity in vivo could not be found.     

The effect of the pneumococcal toxin, pneumolysin on brain ependymal cilia.

Densely ciliated ependymal cells cover the ventricular surface of the brain and cerebral aqueducts separating cerebrospinal fluid, which is infected in meningitis, from neuronal tissue. We have established an ex vivo model that allows measurement of ependymal ciliary beat frequency, using high-speed video analysis, during incubation with bacterial toxins. Ciliated ependyma, from Wistar rats, was exposed to the pneumococcal toxin, pneumolysin, and a mutant form with markedly reduced cytotoxic activity (;0.1%). Wild-type pneumolysin (1500 HU/ml and 150 HU/ml: 10 and 1 microg/ml) caused rapid ciliary stasis (30-150 s), sloughing of cilia and cytoplasmic extrusion. Ciliary slowing before stasis was seen at 15 HU/ml (0.1 microg/ml); however, no effect on ciliary beat frequency was seen at lower concentrations (1.5 HU/ml and 0.15 HU/ml: 0.01 and 0.001 microg/ml). Mutant pneumolysin, 99.9% deficient in haemolytic activity, caused rapid ciliary stasis at 10 microg/ml but no effect was seen at lower concentrations (1-0.1 microg/ml). Pneumolysin, at levels which may be produced during severe pneumococcal meningitis, may cause rapid ependymal ciliary stasis.     

The effect of ovarian steroids on epithelial ciliary beat frequency in the human Fallopian tube

Using a method that detects variations in light intensity we have studied the effect of ovarian steroids on human Fallopian tube epithelial ciliary beat frequency in vitro. We have found that baseline ciliary beat frequency averages between 5-6 Hz. Cilia from ampullary segments of the Fallopian tube beat significantly faster (5.4 Hz+/-0.2) than those from fimbrial segments (4.8 Hz+/-0.2). There was no significant difference in baseline ciliary beat frequency at any other anatomical site in the Fallopian tube. Incubation with progesterone (10 micromol/l) suppresses human Fallopian tube epithelial ciliary beat frequency by 40-50%. This inhibition was observed at similar magnitudes in all Fallopian tubes studied irrespective of anatomical site. Progesterone-induced reductions in ciliary beat frequency were concentration dependent and prevented by the progesterone receptor antagonist mifepristone (RU486). Oestradiol alone (10 micromol/l) had no effect on ciliary beat frequency at any anatomical site in the Fallopian tube but did prevent the reduction in ciliary beat frequency seen with progesterone when tissues were incubated with these two steroids together.      

Ciliary Ultrastructure in Primary Ciliary Dyskinesia and Other Chronic Respiratory Conditions: The Relevance of Microtubular Abnormalities

Twenty-eight subjects with chronic respiratory disease were investigated for clinical data, ciliary beat frequency of nasal mucosa (10 cases), and ciliary ultrastructure. The cases were divided into two groups: those considered compatible with primary ciliary dyskinesia (genetic), and those not fitting into this category (others). A case was defined as genetic if one or more of the following were present: dextrocardia, ciliary beat frequency less than 10 Hz, or an average dynein arm count (outer, inner, or both) of less than two per ciliary cross-section. In each of the genetic cases at least two of these parameters were present. The percentage of malformed microtubules was calculated from the total number of evaluated cross-sections for each case. Ciliary microtubular abnormalities of any kind were no more frequent in cases of primary ciliary dyskinesia than in other cases. The same was true for transposition and radial spoke defects.     

ATP regulation of ciliary beat frequency in rat tracheal and distal airway epithelium

Ciliary beat frequency (CBF) was measured by video-optical microscopy in rat tracheal and distal airway ciliary cells using a slice preparation. In tracheal ciliary cells (tracheal slice), ATP or 2-methylthio ATP (MeSATP) increased CBF, which was inhibited by suramin (100 microm, an inhibitor of purinergic receptor). Ionomycin (5 microm) or thapsigargin (2 microm) increased CBF similarly. Ca2+-free solution or addition of Ni2+ (1 mm) decreased CBF gradually by approximately 25% and subsequent stimulation with ATP (10 microm) increased CBF transiently. The purinergic agonist experiments demonstrated that ATP increases CBF in tracheal ciliary cells via both P2X and P2Y receptors. ATP increased the intracellular calcium concentration ([Ca2+]i) in tracheal ciliary cells. However, in distal airway ciliary cells (lung slice), ATP did not increase CBF and [Ca2+]i, although a Ca2+-free solution decreased CBF, and ionomycin (5 microm) or thapsigargin (2 microm) increased it. Moreover, acetylcholine (100 microm) did not increase CBF in distal airway ciliary cells, although it increased CBF in tracheal ciliary cells. Terbutaline (10 microm), a selective beta2-adrenergic agonist, increased CBF in both tracheal and distal airway ciliary cells. These observations suggest that the Ca2+-mobilization mechanisms via purinergic or muscarinic receptors of the distal airway ciliary cell may be different from those of the tracheal ciliary cell. In conclusion, the CBF increase is differently regulated in the tracheal and distal airway epithelia of the rat.     

Functional characterization of tyrosine transport in fibroblast cells from healthy controls

Ependymal cilia line the ventricular system moving cerebral spinal fluid close to the brain surface. They may be exposed to fluid of increasing viscosity in certain pathological conditions such as bacterial meningitis. Our aim was to determine the effect of increasing viscosity on ciliary function. Ciliated ependyma was exposed to solutions of different viscosities (1-60cP) and ciliary function assessed by high-speed digital imaging. The mean (S.D.) ciliary beat frequency (CBF), measured after 30min incubation in Medium 199 at 37 degrees C, was 34.9 (2.9)Hz. Increased viscous loading was followed by a rapid decrease in CBF compared to baseline readings (p<0.001). After 15min of exposure to the increased viscous load, CBF reached a new stable level while the viscous load was maintained. Compared to baseline measurements of CBF, viscous loading of 3.7cP caused a 16%, 10.4cP at 34% and 24cP a 70% decrease in beat frequency. Further viscous loading at levels up to 60cP resulted in no further reduction of ependymal CBF. Solutions of 24 and 40cP had no effect on ciliary amplitude. An increase in viscosity to 60cP caused a significant (30%: p=0.001) decrease in the ciliary beat amplitude.     

Ciliary beat pattern is associated with specific ultrastructural defects in primary ciliary dyskinesia

BACKGROUND: The main symptoms of primary ciliary dyskinesia (PCD) are nasal rhinorrhea or blockage and moist-sounding cough. Diagnosis can be difficult and is based on an abnormal ciliary beat frequency, accompanied by specific abnormalities of the ciliary axoneme. It is unknown whether determining ciliary beat pattern related to specific ultrastructural ciliary defects might help in the diagnosis of PCD. OBJECTIVE: We sought to determine ciliary beat pattern and beat frequency (CBF) associated with the 5 common ultrastructural defects responsible for PCD. METHODS: Nasal brushings were performed on 56 children with PCD. Ciliary movement was recorded using digital high-speed video imaging to assess beat frequency and pattern. Electron microscopy was performed. RESULTS: In patients with an isolated outer dynein arm or with an outer and inner dynein arm defect, 55% and 80% of cilia were immotile, respectively. Cilia that moved were only flickering. Mean CBF (+/- 95% CI) was 2.3 Hz (+/- 1.2) and 0.8 Hz(+/- 0.8), respectively. Cilia with an isolated inner dynein arm or a radial spoke defect had similar beat patterns. Cilia appeared stiff, had a reduced amplitude, and failed to bend along their length. Immotile cilia were present in 10% of cilia with an inner dynein arm defect and in 30% of radial spoke defects. Mean CBF was 9.3 Hz (+/- 2.6) and 6.0 Hz (+/- 3.1), respectively. The ciliary transposition defect produced a large circular beat pattern (mean CBF, 10.7 Hz [+/- 1.1]). No cilia were immotile. CONCLUSIONS: Different ultrastructural defects responsible for PCD result in predictable beat patterns. Recognition of these might help in the diagnostic evaluation of patients suspected of having PCD.     

Effects of chemical mediators of anaphylaxis on ciliary function

We assessed the effects of selected chemical mediators of anaphylaxis on CBF in vitro. Ciliated epithelial cells were obtained from the trachea of conscious sheep with a cytology brush and suspended in a perfusion chamber containing KH. Ciliary activity was viewed microscopically and recorded on videotape for subsequent slow-motion analysis of CBF. Prostaglandin E1 (10(-8) M to 10(-6) M), prostaglandin E2 (10(-10) M to 10(-6) M), and leukotriene-C4 (10(-8) M) increased CBF between 7% and 33%. Histamine caused ciliostimulation only at the relatively high concentrations above 10(-5) M (7% increase in CBF), whereas prostaglandin F2 alpha (10(-10) M and 10(-6) M) was without effect. In no preparation was ciliary discoordination observed. These findings indicate that several chemical mediators of anaphylaxis stimulate CBF and that the previously described impairment of mucociliary transport in stable allergic asthma or antigen-induced bronchoconstriction is probably not caused by a primary alteration of ciliary function.     

Function and structure of cilia in the fallopian tube of an infertile woman with Kartagener's syndrome

In Kartagener's syndrome (KS), primary defects of the ciliary axoneme cause dyskinetic ciliary motion. Because ciliary motion is an important factor in normal ovum transport, ciliary dyskinesia may cause infertility. On the other hand, the existence of some ciliary activity, albeit abnormal, may account for fertility in some women with KS. In this case study, an infertile woman diagnosed with KS had normal results in all usual infertility tests. Biopsies of tubal mucosa were obtained at laparoscopy for ovum recovery during an in-vitro fertilization cycle. Ciliary activity, measured by laser light- scattering spectroscopy, was detected in all tubal specimens; however the majority of regions sampled showed no activity. In active regions, beat frequency ranged from 5 to 10 Hz, approximately 30% of normal. Electron microscopy showed similar morphological defects in both tubal and nasal mucosa. The number of cilia per cell was approximately 20% of normal. The major ultrastructural abnormality of cilia was an absence of the central microtubules. The only demonstrable explanation for this patient's infertility was primary ciliary dyskinesia associated with KS.      

Adenosine triphosphatase expression correlates with ciliary activity of respiratory epithelium in vitro

In vitro culture of respiratory epithelium is of great utility for pharmacological investigations and tissue engineering. Up to now, the degree of differentiation of respiratory cells cultured in vitro has exclusively been estimated by measuring ciliary beat frequency (CBF). Ciliary motility is dependent on the function of the motor protein dynein that is composed of at least two heavy chains, sharing attributes of adenosine triphosphatases (ATPases). CBF is further dependent on medium conditions and does not allow to draw any accurate conclusion on the proportion of fully differentiated ciliated cells in culture. For this reason we introduced the immunohistochemical detection of a 100-kD ATPase subunit as a correlation with dynein activity in human respiratory cell tissue culture. Our results show that the amount of immunohistochemically detectable ATPase-subunit-positive cells strongly correlates with ciliary motility in vitro. Cultures without ciliary activity exhibited no ATPase staining, whereas in cell cultures with excessive ciliary beat, up to 15.1% of the cells were ATPase positive. Immunohistochemical detection of ATPase in respiratory cell cultures seems to be a sensitive and reproducible complement for the characterization of cultured ciliated epithelium.     

Influence of levocabastine suspension on ciliary beat frequency and mucociliary clearance

The effects of levocabastine, a new fast-acting, highly potent H1-antagonist, on nasal ciliary epithelial function were investigated in an in vitro and in vivo study. In the in vitro study, a suspension of levocabastine in Locke-Ringer solution was applied to 10 bioptic specimens of ciliated human adenoid tissue. Each specimen was exposed to the test solution for 60 min. Ciliary beat frequency (CBF) was recorded with a photoelectric recording device at 10-min intervals. There were small, insignificant decreases in CBF, which were minimal compared to that observed with ciliotoxic agents. In the in vivo study, 8 healthy volunteers were given, intranasally, one droplet of the levocabastine suspension. Mucociliary transit time (MTT) was measured by placing a saccharin particle drenched in indigo carmine in the nose just below the top of the concha and measuring the time until appearance of the dye in the pharyngeal cavity. No statistically significant differences were found in the MTT before and after application of the levocabastine suspension. The studies thus indicate that nasally administered levocabastine does not interfere with ciliary beat frequency and mucociliary function.     

Effect of AA-861, a 5-Lipoxygenase Inhibitor, on Leukotriene Synthesis in Human Polymorphonuclear Leukocytes and on Cyclooxygenase and 12-Lipoxygenase Activities in Human Platelets

AA-861, a selective inhibitor of 5-lipoxygenase of arachidonic acid, was tested for ability to inhibit leukotriene C4 and leukotriene B4 synthesis in human polymorphonuclear leukocytes after calcium ionophore stimulation. AA-861 dose-dependently inhibited leukotriene B4 and leukotriene C4 generation in human polymorphonuclear leukocytes; the concentration required to inhibit generation by 50% (IC50) was 3 X 10(-7) M for leukotriene B4 and 1 X 10(-8) M for leukotriene C4. BW-755C inhibited the generation of leukotriene C4 with an IC50 of about 10(-5) M, indicating that AA-861 is about 1,000 times more potent than BW-755C. AA-861 did not affect the activity of either cyclooxygenase or 12-lipoxygenase at a concentration up to 10(-5) M in human platelets. AA-861 did not inhibit histamine release from human basophils. These results indicate that AA-861 selectively inhibits 5-lipoxygenase but not cyclooxygenase or 12-lipoxygenase in human specimens.     

Presence of Ca2+ is obligatory for the cytotoxic activity of dengue virus-induced cytotoxic factor.

The present study was undertaken to investigate the role of calcium ions (Ca2+) in the cytotoxic activity of the cytotoxic factor (CF) produced by T lymphocytes of the dengue type 2 virus (DV)-infected mouse spleen. It was observed that CF prepared in Ca2(+)-free medium had no cytotoxic activity on normal mouse spleen cells suspended in Ca2(+)-free medium but had activity on cells suspended in medium having Ca2+. The cytotoxic activity of CF was restored by substitution with calcium chloride, the optimal dose being 10(-7) M. CF induced influx of Ca2+ as measured by uptake of radiolabelled calcium chloride (45Ca), in the susceptible target cells, macrophages (M phi) and T lymphocytes, but had no effect on CF-resistant B lymphocytes. Calcium channel blocking drugs, like verapamil, nifedipine and diltiazem, inhibited the cytotoxic activity of CF and also the CF-induced influx of 45Ca in M phi and T cells. Thus, presence of Ca2+ is obligatory for the cytotoxic activity of CF and the cell death is associated with increased intracellular Ca2+.     

Effect of N-acetylcysteine on the ciliary beat frequency of human bronchial explants

The effect of N-acetylcysteine on human bronchial cilia was evaluated by measuring the changes in ciliary beat frequency (CBF) on human bronchial explants exposed to 1%, 2%, 3%, 4%, and 5% concentrations of N-acetylcysteine solution in nutrient medium. There was a progressive reduction in CBF at increasing concentration, indicating an inhibitory effect on human ciliary activity in vitro (P less than 0.05).