Carbon monoxide is endogenously produced in the human nose and paranasal sinuses

Transcription

1 Carbon monoxide is endogenously produced in the human nose and paranasal sinuses Jens A. Andersson, MD, Rolf Uddman, MD, and Lars-Olaf Cardell, MD Malmö, Sweden Background: Carbon monoxide (CO) has recently emerged as an endogenously produced gaseous mediator that, like nitric oxide (NO), appears to be involved in both upper and lower airway inflammation. In healthy subjects a large part of the exhaled NO seems to originate from the nasal airways, and the paranasal sinuses have been described as a dominating site for NO production. Objective: The current study was designed to investigate whether CO could also be produced in the nose and paranasal sinuses. Methods: The occurrence in the nasal mucosa of the enzyme heme oxygenase, the rate limiting step for CO production, was analyzed with use of immunocytochemistry. CO in exhaled and sampled air was measured with an infrared analyzer. Forty-two healthy subjects and two patients with a tracheostoma volunteered for the study. Results: Heme oxygenase like immunoreactivity was seen in the respiratory epithelium, in connection with seromucous glands and in the vascular smooth muscle of the nose. When CO was continuously sampled from one nostril during normal breathing through the mouth, stable levels of CO could be measured within 40 seconds in all subjects tested (n = 33). Repeated measurements indicated only minor variations in the values obtained. Sampling through a drainage tube inserted into the maxillary sinus revealed CO levels comparable to the levels obtained by sampling through the nose (n = 6). Breathing through the nose increased the CO levels obtained in the exhaled air (n = 33, P <.001). Conclusion: These results imply that the nose and paranasal sinuses contribute to the CO production of the human airways. (J Allergy Clin Immunol 2000;105: ) Key words: Carbon monoxide, heme oxygenase, paranasal sinuses There are many sources of endogenous carbon monoxide (CO) production, but the degradation of heme to biliverdin and CO appears to be the dominating one in most species. 1 The enzyme heme oxygenase (HO), with 2 isoforms (HO-1 and HO-2), seems to be the rate-limiting factor. HO-1 is reported to be inducible, whereas HO- 2 is constitutively expressed. 2 HO-2 like immunoreac- From the Department of Otorhinolaryngology, Malmö University Hospital, Malmö, Sweden. Supported by grants from the Swedish Medical Research Council, the Swedish Heart Lung Foundation, the Harald Jeansson Foundation, AGA AB Medical Research Fund, and the University of Lund. Received for publication Aug 9, 1999; revised Oct 21, 1999; accepted for publication Oct 22, Reprint requests: Lars-Olaf Cardell, MD, Department of Otorhinolaryngology, Malmö University Hospital, Malmö , Sweden. Copyright 2000 by Mosby, Inc /2000 $ /1/ Abbreviations used CO: Carbon monoxide DABCO: Triethylenediamine HO: Heme oxygenase NO: Nitric oxide OSA-100: Anti-heme oxygenase-1 polyclonal antibody OSA-200: Anti-heme oxygenase-2 polyclonal antibody tivity is seen in nerve cell bodies in intrinsic ganglia of guinea pig airways and in local parasympathetic ganglia of human trachea and bronchi. These findings suggest that CO serves as a modulator of synaptic neurotransmission in the lung. 3 Both HO-1 and HO-2 like immunoreactivities are also found in the airway smooth muscle and in the respiratory epithelium of guinea pigs, indicating a direct role for CO in airway regulation. 4 The intensity of HO-1 like immunostaining in the lung increases as the result of hypoxic challenge, and exogenous CO is reported to induce a cyclic GMP linked bronchodilation, further supporting the idea of CO as a mediator in airway regulation. 5 Induction of HO-1 by reactive oxygen species followed by increased CO production has been put forward as a general cytoprotective mechanism against oxidative stress and therefore clinically useful in the detection and management of inflammatory lung disorders. 6 This assumption is supported by findings demonstrating increased levels of CO in the exhaled air of asthmatic patients, during periods of nonsteroid treatment, during asthma exacerbation, and as the result of allergen challenge. 7-9 Recent results have indicated that CO, in analogy with nitric oxide (NO), also can be produced in the upper respiratory airways, thus contributing to the total CO content of exhaled air. 10 However, the information in this area is meager and nothing is known about the role of the paranasal sinuses in such a presumed CO production; neither is it currently known how the airway levels of CO are affected by upper airway inflammation. The aims of the current study were to investigate whether the HO enzymes necessary for CO production could be detected in the human nose and to examine whether there is an endogenous production of CO in the nose and the maxillary sinus. METHODS Immunohistochemistry Specimens from human nasal mucosa were obtained in conjunction with nasal surgery (resection of the inferior turbinate) from 5 269

2 270 Andersson, Uddman, and Cardell J ALLERGY CLIN IMMUNOL FEBRUARY 2000 patients (age range 24 to 35 years) with no history of allergic disease. The specimens were immersed in an ice-cold fixative solution composed of 2% formaldehyde and 0.2% picric acid buffered to ph 7.2 with 0.1 mol/l phosphate buffer. After 12 hours the specimens were rinsed for 48 hours in a Tyrode solution containing 10% sucrose, frozen on dry ice, and sectioned in a cryostat at 10-µm thickness. The sections were processed for immunocytochemical studies of the COinducing enzymes HO-1 and HO-2. The antiheme oxygenase-1 polyclonal antibody (OSA-100) and the antiheme oxygenase-2 polyclonal antibody (OSA-200) (Stressgen Biotech, Victoria, Alberta, Canada) were raised in rabbits. The antibodies were used in dilutions of 1:1000 and 1:500, respectively. The sections were exposed to the primary antiserum for 1 to 2 hours in a moist chamber and then rinsed for 10 minutes in PBS. The site of the antigen-antibody reaction was demonstrated after the application of FITC-conjugated antigoat Ig in a dilution of 1:50 for 30 minutes at room temperature. The sections were subsequently mounted in glycerol-pbs with triethylenediamine (DABCO) (Aldrich, Milwaukee, Wis) to prevent fading of the fluorescence. The sections were examined in a Zeiss axioplan system phase/fluorescence microscope. In tissues incubated with primary antiserum (OSA-100) preabsorbed with an excess of recombinant HO-1 (SPP-730, 5 µg/ml) the immunoreactivity was suppressed. Negative controls for nonspecific binding included normal rabbit serum without primary antibody and secondary antibody alone. 4 Because cross-reactions with other proteins containing amino acid sequences recognized by the antisera could not be excluded, it is appropriate to refer to the immunoreactive material as HO- 1 like and HO-2 like. Subjects Healthy nonsmoking volunteers (n = 33, aged 42 ± 3 years, 26 women) not regularly exposed to smoke passively and without continuing medication were investigated to evaluate whether CO could be endogenously produced in the upper airways. All subjects were evaluated by an experienced ear, nose, and throat consultant and subjects with symptoms of respiratory tract infections during the last 3 weeks before entering the study or with a history of allergy, asthma, or sinusitis were excluded. In addition, 2 patients with tracheostoma cannulas as the result of larynx surgery were analyzed separately. In these patients the cannulas prevented exhaled air from reaching the upper airways. Approval for this study was acquired from the local Ethics Committee of the University of Lund and an informed consent was obtained from all subjects. CO measurements CO in exhaled and sampled air was measured with use of an infrared analyzer (Fisher Rosemount NGA 2000, provided by FLS Airloq AB, Stockholm, Sweden). According to the manufacturer, the minimum detectable concentration of CO was 0.2 ppm. The analyzer was calibrated with known concentrations of CO for measurements in the range of 0 to 10 ppm. Gaseous nitrogen (Air Liquide Gas AB, Malmö, Sweden) was used between every set of measurements for baseline verification and 0 calibration. All measurements were made at room temperature (22 C-24 C) in a nonsmoking section of the hospital. Ambient levels of CO were continuously recorded (0.75 ± 0.06 ppm) and were subtracted from measured CO values to compensate for alternating background levels. All subjects were seated in an upright position and all measurements were repeated 3 times with a resting period of 2 to 3 minutes between measurements. CO sampled from the nose A nasal olive was gently introduced into the vestibulum nasi and nasal air was drawn into the CO analyzer by a vacuum pump (0.4 L/min). The contralateral nostril was left open, allowing a stream of room air to enter the nose while the subject was constantly breathing through the mouth. This was the basic sampling technique for CO sampling used throughout the study, when not stated otherwise. Patients with tracheostoma cannulas were analyzed according to the same principles described above. However, these subjects were breathing through their cannulas, which prevented exhaled air from reaching the upper respiratory tract. CO sampled from the maxillary sinus After application of topical anesthesia, a drainage tube was inserted below the inferior nasal turbinate into the right maxillary sinus with use of a SinoJect puncture set (Atos Medical AB, Hörby, Sweden). In a first set of measurements the air in the maxillary sinus was slowly drawn (10 ml/min) into the CO analyzer through the drainage tube. The analyzer was then disconnected from the drainage tube and air was slowly drawn from the contralateral nostril. The drainage tube was sealed until new sets of measurements were performed 16 and 19 hours later. CO in exhaled air The subjects were instructed to breathe normally through the mouth. After 40 seconds they were told to inhale maximally and then to immediately exhale into a 2-L rubber bag (Silkolatex, Willy Rüsch AG, Kernen, Germany). During the whole procedure a nose clamp precluded any passage of air in either direction through the nose. The CO content of the bag was instantly analyzed, with the mean peak value of each set of three measurements used for calculations and statistical analysis. After a resting period the measurements were repeated, this time with the subject breathing and exhaling through the nose, mouth closed. Statistics All results are expressed as means ± SEM. Statistical comparisons were made by the Student t test and P values <.05 were considered statistically significant. RESULTS In the nasal turbinate HO-2 and HO-1 like immunoreactivities were observed in the respiratory epithelium and in connection with the smooth muscle of arteries and veins in the submucosa, as well as in association with seromucous glands. The HO-2 staining was stronger than the HO-1 staining of corresponding structures. Nevertheless, HO-1 immunostaining was consistently present in all turbinates investigated (Fig 1). When CO was continuously sampled from one nostril during normal breathing through the mouth, stable CO levels of 1.80 ± 0.06 ppm were reached within 40 seconds (n = 33). During this procedure sampled air was continuously replaced by a mixture of room air, through the contralateral nostril, and air from the lower airways. Repeated measurements indicated only minor variations in the obtained values (3 consecutive measurements revealed a variation of 5.3% ± 0.8% in relation to the mean value) (Fig 2). There was no difference in the values obtained from the left and right nostrils in the same patient. Nasal breathing resulted in higher CO levels in the exhaled air compared with breathing only through the mouth (2.37 ± 0.07 ppm and 2.20 ± 0.05 ppm, respec-

3 J ALLERGY CLIN IMMUNOL VOLUME 105, NUMBER 2, PART 1 Andersson, Uddman, and Cardell 271 FIG 1. Localization of HO-2 and HO-1 immunoreactivity in human nasal mucosa. A, Strong HO-2 immunostaining in smooth muscle of small artery. B, HO-2 immunostaining of surface epithelium and seromucous glands. C, Weak HO-1 immunostaining of smooth muscle in artery. D, HO-1 immunostaining of surface epithelium. (Original magnification, 300.) FIG 2. Nasal CO levels continuously sampled with nasal olive in vestibulum nasi. Results from 6 consecutive measurements from each subject, alternating between the left and right nostril. Data are mean ± SEM, n = 33. tively, n = 33, P <.001, Fig 3). CO levels obtained in the exhaled air were generally higher than CO levels obtained with use of the continuous sampling technique (n = 33, P <.001). This was a result of the addition of ambient air through the contralateral nostril resulting in a dilution of the gases produced in the nose during the latter technique. During slow sampling of air from the maxillary sinus and the nasal cavity a plateau phase for the registered CO levels was reached within 3 to 4 minutes (because of the

4 272 Andersson, Uddman, and Cardell J ALLERGY CLIN IMMUNOL FEBRUARY 2000 FIG 3. CO levels measured in air exhaled through mouth (open column) and nose (solid column) after 40 seconds of breathing through mouth and nose, respectively. Data are mean ± SEM, n = 33. Three asterisks, P <.001. A dead space of the setup) for both types of measurements. These levels remained stable during the rest of the sampling. The CO levels derived from the maxillary sinus during the first set of analyses did not differ from the levels obtained in the nasal cavity (1.59 ± 0.10 ppm and 1.60 ± 0.25 ppm, respectively, n = 5) (Fig 4, A). When the procedure was repeated after 16 and 19 hours, the same principal results were obtained (Fig 4, B). In nasal air sampled from patients with tracheostoma cannulas representing a permanent closure of the connection between upper and lower airways, stable levels of CO could be readily measured (1.31 ± 0.20 ppm, n = 2). DISCUSSION The current study demonstrates that CO can be reproducibly measured in the nose and paranasal sinuses and that enzymes responsible for local CO production are present in the nasal airways. There is some evidence that increased CO levels in exhaled air may reflect lower airway inflammation in asthmatic patients. 7-9 However, the source of CO within the lungs is unknown and the role of CO in the upper airways has previously not been evaluated. A relationship between HO activity and CO production has been demonstrated in several types of peripheral tissues, 11,12 and the presence of HO immunoreactivity in the nose, reported in this study, indicates a local CO production in the upper respiratory airways. Weak HO-1 and strong HO-2 like immunoreactivity has, in analogy with the current findings, recently been reported in the lower respiratory tract of guinea pigs. 4 Furthermore, the HO-1 B FIG 4. A, Time course for CO levels in air continuously sampled from nose (solid squares) and through draining tube inserted into right maxillary sinus (open circles). Data are mean ± SEM, n = 5. B, Time course for CO levels in air continuously sampled from left nostril (solid symbols) and from maxillary sinus (open symbols). Measurements were repeated 3 times within 24 hours (first measurement, circles; repeated after 16 hours, squares; repeated after 19 hours, triangles). Drainage tube was sealed between measurements. Data are mean ± SEM, n = 5.

5 J ALLERGY CLIN IMMUNOL VOLUME 105, NUMBER 2, PART 1 Andersson, Uddman, and Cardell 273 immunoreactivity in the guinea pig lung was shown to increase as the result of hypoxic challenge, indicating the ability to up-regulate endogenous CO production in the airways. Although the HO-1 and HO-2 enzymes seem to have an equal ability to induce CO production, they have little in common in primary structure, regulation, or tissue distribution, and they are derived from different genes. 13 A role for CO as a peripheral transmitter involved in nonadrenergic, noncholinergic relaxation has been proposed, 14 and recent in vivo results indicate that exogenous CO can induce bronchodilation by an NOindependent, cyclic GMP related mechanism. 5 Measurements of CO in exhaled bagged air is often used to relate CO levels in the human airways to functional and inflammatory events. 15 However, this mainly reflects events in the lower respiratory tract. In the current study we describe a method for continuous CO sampling in the nose. This method is well reproducible, easy to use, requires a limited patient cooperation, and provides a good reflection of events also in the upper respiratory tract. NO, a constituent of normal exhaled human breath, is a well-established endogenous vasodilator and inflammatory mediator. 16,17 A large portion of NO found in the human breath derives from the upper airways, 18,19 where the paranasal sinuses appear to be the dominant site of production. NO concentrations have been reported to be 10 times higher in the sinuses than in the nose. 20 To investigate whether high concentrations of CO are also found in the paranasal structures, direct measurements were made from the maxillary sinus. The same concentration of CO was found in the maxillary sinus as in the nose, indicating a uniform production in nasal and paranasal structures. Breathing through the nose increased the amount of CO found in exhaled air, pointing to a contribution from the upper airways to the total CO production within the airways. Further evidence was derived from the nasal CO measurements of 2 patients with tracheostoma cannulas in which there was no exchange of air between the upper and lower respiratory tract. In conclusion, HO-2 and HO-1 like immunoreactivities were observed both in the respiratory epithelium and in connection with the smooth muscle of arteries and veins in the submucosa. It is tempting to assume that the epithelium is the major site of production for the CO found in the nose and paranasal sinuses, although a contribution from the underlying mucosa and smooth muscle cannot be excluded. Further studies will be necessary to investigate whether CO produced in the upper airways, in analogy with NO, has a role in human airway regulation and inflammatory response. REFERENCES 1. Rodgers PA, Vreman HJ, Dennery PA, Stevenson DK. Sources of carbon monoxide (CO) in biological systems and applications of CO detection technologies. Semin Perinatol 1994;18: Maines MD, Trakshel GM, Kutty RK. Characterization of two constitutive forms of rat liver microsomal heme oxygenase: only one molecular species of the enzyme is inducible. J Biol Chem 1986;261: Canning BJ, Fischer A. Localization of heme oxygenase-2 immunoreactivity to parasympathetic ganglia of human and guinea-pig airways. Am J Respir Cell Mol Biol 1998;18: Cardell LO, Lou YP, Takeyama K, Ueki IF, Lausier J, Nadel JA. Carbon monoxide, a cyclic GMP-related messenger, involved in hypoxic bronchodilation in vivo. Pulm Pharmacol Ther 1998;11: Cardell LO, Ueki IF, Stjarne P, Agusti C, Takeyama K, Linden A, et al. Bronchodilatation in vivo by carbon monoxide, a cyclic GMP related messenger. Br J Pharmacol 1998;124: Horvath I, Loukides S, Wodehouse T, Kharitonov SA, Cole PJ, Barnes PJ. Increased levels of exhaled carbon monoxide in bronchiectasis: a new marker of oxidative stress. Thorax 1998;53: Zayasu K, Sekizawa K, Okinaga S, Yamaya M, Ohrui T, Sasaki H. Increased carbon monoxide in exhaled air of asthmatic patients. Am J Respir Crit Care Med 1997;156: Yamaya M, Sekizawa K, Ishizuka S, Monma M, Sasaki H. Exhaled carbon monoxide levels during treatment of acute asthma. Eur Respir J 1999;13: Paredi P, Leckie MJ, Horvath I, Kharitonov SA, Barnes PJ. Exhaled carbon monoxide is elevated during allergen challenge in asthmatic patients. Eur Respir J 1998;28(Suppl): Yamaya M, Sekizawa K, Ishizuka S, Monma M, Mizuta K, Sasaki H. Increased carbon monoxide in exhaled air of subjects with upper respiratory tract infections. Am J Respir Crit Care Med 1998;158: Maines MD. Heme oxygenase: function, multiplicity, regulatory mechanisms, and clinical applications. FASEB J 1988;2: Vreman HJ, Stevenson DK. Heme oxygenase activity as measured by carbon monoxide production. Anal Biochem 1988;168: Cruse I, Maines MD. Evidence suggesting that the two forms of heme oxygenase are products of different genes. J Biol Chem 1988;263: Rattan S, Chakder S. Inhibitory effect of CO on internal anal sphincter: heme oxygenase inhibitor inhibits NANC relaxation. Am J Physiol 1993;265:G Jarvis MJ, Russell MA, Saloojee Y. Expired air carbon monoxide: a simple breath test of tobacco smoke intake. BMJ 1980;281: Gustafsson LE, Leone AM, Persson MG, Wiklund NP, Moncada S. Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. Biochem Biophys Res Commun 1991;181: Borland C, Cox Y, Higenbottam T. Measurement of exhaled nitric oxide in man. Thorax 1993;48: Gerlach H, Rossaint R, Pappert D, Knorr M, Falke KJ. Autoinhalation of nitric oxide after endogenous synthesis in nasopharynx. Lancet 1994;343: Lundberg JO, Weitzberg E, Nordvall SL, Kuylenstierna R, Lundberg JM, Alving K. Primarily nasal origin of exhaled nitric oxide and absence in Kartagener s syndrome. Eur Respir J 1994;7: Lundberg JO, Farkas-Szallasi T, Weitzberg E, Rinder J, Lidholm J, Anggaard A, et al. High nitric oxide production in human paranasal sinuses. Nat Med 1995;1:370-3.