Effects on the ciliated epithelium of protein D-producing and -nonproducing nontypeable Haemophilus influenzae in nasopharyngeal tissue cultures.

A pair of isogenic, nontypeable Haemophilus influenzae strains, one expressing protein D and the other protein D-negative, was compared in their ability to cause damage in a human nasopharyngeal tissue culture model. Damage was assessed by measuring the ciliary beat frequency (CBF) of tissue specimens at 12 h intervals. Cultures inoculated with H. influenzae manifested a decrease in CBF beginning after 12 h, with a maximum decrease after 36 h. The impairment of ciliary function by the protein D-expressing strain was significantly greater than that caused by the protein D-negative mutant (P<.01). Tissue specimens examined by scanning and transmission electron microscopy after 24 h appeared normal. After 48 h of incubation, the protein D-expressing strain caused a significant loss of cilia. These findings suggest that protein D is involved in the pathogenesis of upper respiratory tract infections due to nontypeable H. influenzae, probably by enhancing functional and morphological damage to cilia.

Ciliostimulatory effects mediated by nitric oxide.

Ciliostimulation induced by various transmitters has been suggested to be mediated by the release of nitric oxide (NO). Freshly obtained adenoid tissue explants were pre-treated with the nitric oxide synthase (NOS) inhibitor N(G)-nitro L-arginine (L-NNA), to determine whether the ciliostimulators terbutaline, methacholine, substance P, and endothelin-1 require the release of NO to increase ciliary beat frequency (CBF) in vitro. The L-NNA pre-treatment affected the change in CBF induced by each of the ciliostimulators tested. To determine whether cyclic nucleotides also stimulate CBF by inducing the release of NO, an extra series of experiments were performed with dibutyryl cAMP and dibutyryl cGMP, and L-NNA pre-treatment. In contrast to the experiments with the various ciliostimulators, both dibutyryl cAMP and dibutyryl cGMP exerted ciliostimulatory effects that could not be inhibited by L-NNA. The present findings suggest that NO acts as an intermediate messenger in the ciliated epithelium in response to various transmitters and mediators. On the other hand, pre-treatment with the NOS inhibitor L-NNA did not affect ciliary response to the second messengers cAMP and cGMP, thus suggesting that NO dependent mechanisms do not constitute the sole pathway for the stimulation of ciliary function.

Nitric oxide is a regulator of mucociliary activity in the upper respiratory tract.

The in vitro effects of the nitric oxide (NO) substrate L-arginine on ciliary beat frequency and the in vivo effects of the NO donor sodium nitroprusside (SNP) on mucociliary activity were investigated in the rabbit maxillary sinus mucosa with photoelectric techniques. L-Arginine increased ciliary beat frequency in vitro with a maximum response of 27.1% +/- 6.4% at 10(-3) mol/L, and this effect was reversibly blocked by pretreatment with the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine, whereas D-arginine had no such effect. SNP increased mucociliary activity in vivo, the peak response of 36.8% +/- 4.2% being obtained at the dose of 30.0 microg/kg. No tachyphylaxis was observed after repeat challenge with SNP. The increase in mucociliary activity caused by SNP was largely unaffected by pretreatment with the calcium channel blocker nifedipine, the cyclooxygenase inhibitor diclofenac, and the cholinergic antagonist atropine. The nonselective beta-blocker propranolol delayed the peak response of SNP to 7 to 8 minutes after challenge, compared with 1 to 2 minutes after challenge in animals without pretreatment. The results show the NO substrate L-arginine and the NO donor SNP to have ciliostimulatory effects in vitro and in vivo, respectively. The occurrence of NOS production in the sphenopalatine ganglion and sinus mucosa of the rabbit was studied by immunohistochemistry for NOS activity or nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry. The latter is an indirect sign of neuronal NOS activity. Numerous NOS-containing cell bodies were seen in the sphenopalatine ganglion; in the sinus mucosa a moderate supply of thin NOS-immunoreactive nerve fibers was seen. Taken together, the morphologic findings and the functional results indicate NO to be a regulator of mucociliary activity in upper airways.

Effects of nitric oxide on blood flow and mucociliary activity in the human nose.

In an animal model, nitric oxide (NO) has been shown to increase mucociliary activity in vivo and ciliary beat frequency in vitro. The aim of the present study was to investigate the effects of NO on blood flow and mucociliary activity in the human nose. The concentration of NO in nasal air was measured with a chemiluminescence technique after nebulizing the NO donor sodium nitroprusside (SNP) at a dose of 3.0 mg into the nose in six volunteers, and was found to increase by 50.1% +/- 10.0% (mean +/- SEM; p < .001) after the SNP challenge. Blood flow measured by laser Doppler flowmetry increased by 67.3% +/- 15.5% (p < .05) after challenge with SNP at 1.0 mg, and by 75.4% +/- 18.5% at 3.0 mg (p < .01; n = 6). The higher dose, which produced no subjective side effects, was then used in the mucociliary experiments. The maximum increase in nasal mucociliary activity was 57.2% +/- 6.7% at 3.0 mg of SNP (n = 5). The findings support the view that NO regulates mucociliary activity and blood flow in the human nasal mucosa.

Low levels of nasal nitric oxide (NO) correlate to impaired mucociliary function in the upper airways.

Findings in previous studies have suggested nitric oxide (NO) to be a regulator of mucociliary activity in the upper airways. The aim of the present investigation was to study whether a correlation exists between the nasal NO concentration and mucociliary function in patients suffering from respiratory tract diseases such as chronic sinusitis or recurrent pneumonia. Nasal NO was measured with a chemiluminescence analyser, 100 ppb (parts per billion) being adopted as the lower limit of the normal range on the basis of findings in an earlier study of healthy subjects. Mucociliary function was evaluated by measurements of ciliary beat frequency (CBF) in nasal brush samples, and the saccharin transport test. A subnormal level of nasal NO was found in 50% (9/18) of the patients. This correlated with a significantly impaired mucociliary function, regarding both CBF and the saccharin transport time. The median CBF was 10.6 Hz in the group with normal levels of nasal NO, as compared to 8.4 Hz in the subnormal NO group. All patients with a normal nasal NO concentration had a mean CBF of > or = 9.0 Hz in their nasal brush samples, but in the subnormal group the same measurements yielded a CBF of > or = 9.0 Hz in only 22% (2/9) of the cases. As measured with the saccharin test, mucociliary transport was normal in 78% (7/9) in the normal nasal NO group, but the saccharin test was normal only in 11% (1/9) of the subnormal nasal NO group. Nasal NO levels were found to correlate with both CBF measurements (Spearman's rho, 0.80) and the saccharin transport test results (Spearman's rho, -0.61). The results of the present study provide further support for the view that NO is an important regulator of mucociliary function in the upper airways, and that measurements of the nasal NO concentration should be included in investigations of the mucociliary system.

Nitric oxide (NO) production in the upper airways is decreased in chronic sinusitis.

The nasal concentration of nitric oxide (NO) was measured by chemiluminescence in healthy volunteers 3-68 years of age, and in patients suffering from common cold and chronic sinusitis. The concentration of NO in healthy subjects, 233.2 +/- 66.8 ppb (mean +/- SD), was found to be relatively independent of age and body size. The measured levels of NO did not differ between healthy volunteers and common cold patients, but they were significantly lower in patients suffering from chronic sinusitis, 96.4 +/- 72.8 ppb. As NO is a regulator of mucociliary activity and has bacteriostatic and antiviral effects, the decreased concentration of NO in patients suffering from sinusitis suggests that lack of NO may contribute to the pathogenesis of this disease. The importance of NO for the mucociliary system was emphasized by the finding that the 2 patients with the lowest nasal concentration of NO were found to manifest functional and morphological changes of the mucociliary system that are typical of acquired mucociliary dysfunction.

Recordings of mucociliary activity in vivo: benefit of fast Fourier transformation of the photoelectric signal.

Investigations of mucociliary activity in vivo are based on photoelectric recordings of light reflections from the mucosa. The alterations in light intensity produced by the beating cilia are picked up by a photodetector and converted to photoelectric signals. The optimal processing of these signals is not known, but in vitro recordings have been reported to benefit from fast Fourier transformation (FFT) of the signal. The aim of the investigation was to study the effect of FFT for frequency analysis of photoelectric signals originating from an artificial light source simulating mucociliary activity or from sinus or nasal mucosa in vivo, as compared to a conventional method of calculating mucociliary wave frequency, in which each peak in the signal is interpreted as a beat (old method). In the experiments with the artificial light source, the FFT system was superior to the conventional method by a factor of 50 in detecting weak signals. By using FFT signal processing, frequency could be correctly calculated in experiments with a compound signal. In experiments in the rabbit maxillary sinus, the spontaneous variations were greater when signals were processed by FFT. The correlation between the two methods was excellent: r = .92. The increase in mucociliary activity in response to the ciliary stimulant methacholine at a dosage of 0.5 microgram/kg was greater measured with the FFT than with the old method (55.3% +/- 8.3% versus 43.0% +/- 8.2%, p < .05, N = 8), and only with the FFT system could a significant effect of a threshold dose (0.05 microgram/kg) of methacholine be detected. In the human nose, recordings from aluminum foil placed on the nasal dorsum and from the nasal septa mucosa displayed some similarities in the lower frequency spectrum (< 5 Hz) attributable to artifacts. The predominant cause of these artifacts was the pulse beat, whereas in the frequency spectrum above 5 Hz, results differed for the two sources of reflected light, the mean frequency in seven healthy volunteers being 7.8 +/- 1.6 Hz for the human nasal mucosa. It is concluded that the FFT system has greater sensitivity in detecting photoelectric signals derived from the mucociliary system, and that it is also a useful tool for analyzing the contributions of artifacts to the signal.

Effects of endothelins 1, 2 and 3 on mucosal blood flow in the upper airways.

The three endothlins 1, 2 and 3 (ET-1, ET-2 and ET-3) are 21-amino-acid peptides which in a previous study in the rabbit have been shown to increase both mucociliary activity in the maxillary sinus in vivo and ciliary beat frequency (CBF) in sinus and tracheal samples in vitro. We have also demonstrated positive immunohistochemical staining for endothelin in the epithelium of samples from both maxillary sinus and trachea. The aims of the present investigation were to study the effects of the three endothlins on mucosal blood flow in the maxillary sinus of rabbits and the nasal mucosa of humans. ET-1, ET-2 and ET-3 decreased mucosal blood flow in the maxillary sinus of rabbits measured in vivo with laser Doppler flowmetry (LDF). Significant dose-response relationships were established for all endothelins. A comparison of the regression lines of the dose-response relationships for ET-1 and ET-2 did not show any difference, whereas the regression line of the dose-response relationships for ET-3 differed significantly. Maximum effect was seen at 50.0 pmol/kg ET-1, which reduced blood flow by -74.4 +/- 7.0%. Vasoconstriction became evident 30 s after administration of the endothelins, reached its maximum after 1 min and lasted 10 to 20 min. ET-1 induced stronger vasconstriction than ET-2 and ET-3 at equal dosage, except at 100.0 pmol/kg, where ET-2 had the same effect as ET-1. No tachyphylaxis was observed after repeated 10.0 pmol/kg-dose of ET-1. Similarly, there was no tachyphylactic effect on mucociliary activity in vivo in the maxillary sinus of rabbits after repeated challenges with ET-1 at 10.0 pmol/kg. Pretreatment with the cyclooxygenase inhibitors diclofenac had no inhibitory effect on the vasoconstriction induced by ET-1 at 10.0 and 50.0 pmol/kg. In 6 human volunteers the peak blood flow in the nasal septum decreased by -41.8 +/- 11.6% (p < 0.05) and AUC (area under curve) values decreased by -612.0 +/- 322.4% (p < 0.05) after challenge with 0.1 nmol ET-1, delivered in aerosol form into the nose. The effects of the endothelins on the blood flow in the upper airways of rabbit and man indicate that they have a functional role in the regulation of the mucosal vasculature of the upper airways.

The effects of formoterol, a long-acting beta 2-adrenoceptor agonist, on mucociliary activity.

The effect on mucociliary function of formoterol, a beta 2-adrenoceptor agonist bronchodilator with a prolonged duration of action (as compared with salbutamol or terbutaline), was investigated both in vitro and in vivo with a photoelectric technique. Formoterol, and its (R,R)-enantiomer, increased ciliary beat frequency in vitro in guinea pig trachea preparations (peak increase 17.2 +/- 2.0% at a concentration of 10(-7) M) and in vivo in the rabbit maxillary sinus (peak increase 23.0 +/- 4.0% at a dosage of 1 nmol/kg). Formoterol was approximately 100 times more potent than terbutaline in vitro, as judged from the dose-response curve. The main difference between their effect in vivo was the 2-fold longer duration of the mucociliary acceleration after formoterol at 1 nmol/kg than after terbutaline at the equi-effective dosage of 10 nmol/kg terbutaline (20 vs. 10 min, respectively). The findings indicate formoterol to be a powerful, long-acting ciliostimulant, a property which may be of clinical advantage in the treatment of airway disease.

Method for in vivo measurement of mucociliary activity in the human nose.

The aim of the investigation was to develop a method for observing mucociliary activity in the human nose in vivo without possible artifacts introduced by anesthesia or surgical intervention. A probe containing an optical system was constructed for the purpose, mucociliary activity on the nasal septum being photoelectrically analyzed by computer. Challenges with pharmacologic substances were administered with a standardized nebulizer delivering an aerosol heated to 33 degrees C. The baseline mucociliary wave frequency in vivo was 691.7 +/- 93.0 waves per minute (11.5 +/- 1.6 Hz), and the corresponding ciliary beat frequency in vitro was 800.7 +/- 85.4 beats per minute (13.3 +/- 1.4 Hz). The coefficients of variation were 13.4% between individuals and 11.3% +/- 2.1% (range 8.3% to 14.3%) within a subject. The method showed good reproducibility regarding recordings from different spots on the mucosa and on a day-to-day basis. Challenge with the beta 2-agonist terbutaline sulfate produced an increase of mucociliary activity of 40.6% +/- 7.8% (mean +/- SEM), which is consistent with previous results in animal models. This is the first report of a method suitable for in vivo studies of the mucociliary effects of challenge with autonomic agonists and airway irritants in humans.

Mucociliary function and endothelins 1, 2, and 3.

Endothelins are recently discovered peptides that cause vasoconstriction and bronchoconstriction. The significance to the mucociliary system of endothelins (ET) 1, 2 and 3 in upper as well as lower airways has not yet been clarified. Effects of these active peptides were investigated, combining both in vitro and in vivo rabbit models of mucociliary activity from the maxillary sinus and the trachea. The studies were performed using computerized photometric microscopy. Immunohistologic staining procedures were used to determine the presence of endothelins in sinus and trachea epithelium. Significant effects on mucociliary activity were noted for ET-1, -2, and -3 in vitro as well as in vivo. All endothelins were noted to accelerate mucociliary activity of both sinus and tracheal mucosa. The effects of endothelins were greater in the sinus than in the trachea. In vitro studies using the calcium blocker nifedipine and the cyclooxygenase inhibitor diclofenac indicated that the mechanism of action involves an intermediary prostaglandin pathway but is independent of release of intracellular calcium. These results were confirmed using ET-1 in vivo. Immunohistochemical staining showed endothelin to be present in both maxillary and tracheal epithelium of the rabbit. We conclude that endothelins have significant mucociliary stimulatory effects and that the presence of endothelins in normal mucosa indicates a potentially important role in respiratory homeostasis as well as inflammatory conditions.