Respiratory Nitric Oxide Levels in Experimental Human Influenza*

Transcription

1 Respiratory Nitric Oxide Levels in Experimental Human Influenza* Andrew W. Murphy, MD; Thomas A.E. Platts-Mills, MD, PhD; Monica Lobo, MD; and Frederick Hayden, MD Background: Exhaled oral nitric oxide (NO), a reported marker of inflammation in the respiratory tract, can be elevated by "upper respiratory tract infections." However, the responsible viruses and the time course of this rise in NO are not clear. Objective: To determine the expired nasal and oral NO levels during experimentally induced influenza A infection in 14 healthy volunteers without a history of asthma, rhinitis, or sinusitis. Methods: After being housed in individual rooms, susceptible volunteers were inoculated with 1 6 5% tissue culture infective dose of influenza Affexas/36/91/(H1N1) virus on a single occasion by intranasal drops. Volunteers remained in the isolation unit for 8 days and returned for follow-up 21 days after inoculation. Symptom scores and nasal washing for viral culture were obtained daily. NO samples from the mouth and nose were obtained on days through 4, 8, and 21 by having the patient perform a slow vital capacity maneuver through a plastic tube into a Mylar balloon. Results: All patients had influenza virus cultured from nasal washings (12 of 14 on day 1, 14 of 14 by day 5). Patient symptom scores peaked on day 3 (mean±se; 15.4±3.2) and returned to baseline by day 8. Preinfection exhaled nasal NO (right, 28.4±3.7 parts per billion [ppb]; left, 27.7±4.6 ppb) was significantly higher than oral NO (5.8 ppb; p<.1). Exhaled oral NO was significantly elevated on day 8 postinoculation (12.9±.8 ppb; p<.1 Bonferroni) and returned to baseline at follow-up. Nasal NO levels showed a slight decrease on days 2 to 4 but returned to baseline by day 8. Conclusion: Experimental influenza virus infection can increase oral but not nasal exhaled NO levels. The timing of exhaled NO changes suggests that NO does not contribute to illness manifestations directly. (CHEST 1998; 114: ) Key words: influenza A; nitric oxide; upper respiratory trad infection Abbreviations: MDCK Madin Darby canine kidney; NOnitric oxide; ppbparts per billion Nitric oxide (NO) is an endogenously produced molecule that may play an important role in airway pathophysiology. NO, which is produced via a stereospecific reaction from L-arginine through the enzyme NO synthase (NOS), 1 can be measure in the exhaled oral and nasal air of humans. 2 - L 2 Recently, it has been suggested that orally exhaled NO can be an important marker of inflammation in asthma and is most likely not the result of contamination from the nasopharynx Treatment of asthmatics with *From the Asthma and Allergic Diseases Center (Drs. Murphy and Platts-Mills), Division of Epidemiology and Virology (Dr. Hayden), Departments of Medicine (Drs. Murphy, Platts-Mills, and Hayden), Pathology (Dr. Hayden), and Student Health (Dr. Lobo), University of Virginia School of Medicine, Charlottesville. Supported in part by an unrestlicted grant from Biota Holdings Ltd, Melbourne, Australia, and by NIH grant 1U1-AI Manuscript received August 29, 1997; revision accepted Februaly 6, glucocorticoids can decrease the measured NO levels,5,?,8 suggesting that inducible NOS is responsible for the elevated NO levels seen in asthma. Exhaled oral NO can be elevated in those with seasonal allergic rhinitis 9 and during upper respiratory tract illnesses of undefined etiology.l 2 These observations suggest that exhaled oral NO can be a sensitive marker of inflammation in the respiratory tract, but not specific for the inflammation seen in asthma. Finally, exhaled nasal NO levels have been found to be higher than oral NO levels 9 11 and elevated from baseline in those with seasonal allergic rhinitisy Previous work has suggested that influenza and other respiratory viruses, which can result in the increased production of inflammatory cytokines, may play an important role in the pathogenesis of asthma.l3-17 Although previous evidence has shown that patients who present with "upper respiratory tract" infections have increased exhaled oral NO, the vi- 452

2 Table l-tcid 5 for Virus Collection Methods* Days Postinoculation Specimen Day 1 Day 2 Day 3 Nasal wash 2.±.4 3.8±.5 3.2±.5 Nasal swab.5±.2 1.4±.3 1.±.2 Throat gargle.6±.3 2. ±.5 1.4±.3 Throat swab.5±.2 1.±.2.5±.2 *TCID 5 tissue culture infective dose at 5% (logh/ml). ruses responsible for the increased NO seen were not determined and the time course of the elevation was not studied. To investigate the relationship between viral infection and exhaled NO, we have followed exhaled nasal and oral NO in healthy volunteers experiencing experimentally induced influenza infection. Viral Inoculation MATERIALS AND METHODS The study was an open-labeled, nonrandomized trial conducted in June Fourteen healthy male or nonpregnant female volunteers who did not have a histmy of asthma, allergic rhinitis, or sinusitis were recruited for participation based on suseeptibility to the challenge virus (hemagglutination-inhibition antibody titers :S 1:8). None of the subjects were taking inhaled or oral corticosteroids, theophylline preparation, or antihistamines prior to enrollment. On the night before viral inoculation, the subjects were confined to an isolation unit and housed in individual rooms for 8 days. The following moming the subjects were inoculated intranasally with approximately 1 6 5% tissue culture infective dose of influenza A!fexas/36/91 (H1N1 ) virus (provided by Dr. L. Potash; Program Resources Inc; McLean, Va) on a single occasion by intranasal drop (.25 ml to each nostril). Viral and Clinical Monitoring Nasal washings were collected before viral inoculation for deteetion of wild-type virus infection by standard culture techniques. Thereafter, nasal washings (1 ml) and throat gargle (1 ml) samples were collected daily for the recovery of influenza virus using Maclin Darby canine kidney (MDCK) cells. Samples yielding virus on initial culture in MDCK cells were titered in monolayers of MOCK cells by using se1i al1-fold dilutions after single fi eeze thaw of the original sample. Patient symptom scores were assessed in the morning and aftemoon of days through 8. Subjects were asked to rate on a four-point scale (Onone, 1mild, 2moderate, 3severe) each of the following: nasal stuffiness, earache/pressure, runny nose, sore throat, cough, sneezing, breathing difllculty, myalgia, fatigue, headache, feverish feeling, hoarseness, chest discomfort, and overall discomfort. Oral temperatures were recorded twice daily. A continued oral temperature >37.7 C was considered to indicate fever. Measurement of NO Samples for NO measurement were taken on day (before challenge) and days l, 2, 3, 4, 8, and 21 postinoculation. Samples Day 4 Day 5 Day 6 Day 7 Day 8 2.7±.4 2.6±.5 1.4±.5.6±.3.3±.2 1.±.3 1.1±.3.6±.2.3±.2.2±.1.9±.3.9±.3.6±.3.1±.1.3±.2.2±.1.1±.1.3±.1.1±.1.±. for NO were taken by having the subject perform a slow vital capacity maneuver (over 1 to 15 s) through a plastic tube into a Mylar balloon as previously desc1ibed.s When sampling from the mouth, patients inspired to maximal inspiration and then placed the plastic tube into their mouth. After occluding both nostiils, they exhaled through the plastic tube into the Mylar balloon. When sampling from the nostril, the patient inserted the plastic tube in the sampling nostril, inspired to maximal inspiration, occluded the nonsampled nostril, and then performed the slow vital capacity maneuver through the nostril. The contents of the balloons were analyzed for NO, within 2 h, via a chemiluminescence reaction by an analyzer (Siever 28 NO Analyzer; Sievers Corp; Boulder, Colo). All values for NO are reported as parts per billion (ppb). After allowing the NO analyzer to equilibrate with each individual sample for 45 to 6 s, data were acquired at a rate of one sample per second over 1 s. The average of this 1-s sample was calculated and recorded as the value for that particular sample. The NO analyzer was calibrated at the beginning of the study, and then daily, with ppb NO and 9.9 ppm NO gas standards (BOC Gases; Charlottesville, Va). Exhaled mixed oral NO was measured each of the experimental days from a control patient and did not reveal any significant difle rence (values are in ppb: 6.6, 6., 5.8, 6.2, 8., 7.5, and 6.9). To ensure that the Mylar balloons did not absorb NO from the ambient air, a Mylar balloon was filled with the ppb gas standard and sampled at, 1, and 2 h on each day of sample measurement. A corrected patient value for NO was calculated by subtracting the value recorded for the ppb balloon control from the patient sample (range, to.8 ppb). Data Analysis NO concentrations are reported in ppb and the results are expressed as mean±se. Patient symptom scores are expressed as (!) 1 6 * * 1 4 s 1 r:/) s.. 8 >.. r:/) Days Post - Inoculation FIGURE 1. Patient total symptom score vs time postinoculation. Data points are the mean ±SE. Comparison for any particular day is made to day (asterisk: p <.1 by Bonferroni multiple comparison). CHEST 1114 I 2 I AUGUST,

3 the mean::'::se. Comparisons of NO levels and patient symptom scores on different days were made by analysis of variance. When differences were detected, they were further evaluated by multiple comparisons of the mean. Patient 8 did not provide any data on day 8 of the study and was excluded from analysis on this day. Patient 4 on day 3 postinoculation could not provide an NO sample from her left nostril and on day 4 could not provide an NO sample from her right nostril. These data points were not included in that day's analysis. RESULTS Infection and Illness Occurrence Fourteen patients (1 men: mean age, 26.1 years; range, 18 to 36 years; and 4 women: mean age, 23.8 years; range, 18 to 4 years) participated in this study. None of the subjects had virus isolated prior to inoculation and all became infected afterwards. Following challenge, 12 subjects (86%) had positive viral culture on the first day after inoculation. For two volunteers (subjects ll and 14), viral cultures became positive on days 4 and 5, respectively. All but two volunteers were negative for virus at both sites by day 8. The highest viral titers were obtained from nasal washings and peaked on day 2 postinoculation (Table l). Ten (71%) subjects developed upper respirat1y tract infection symptoms, five (36%) had cough on ;::::2 days, seven (5%) had fever on at least 1 day, and nine (64%) volunteers had myalgias on ;::::2 days. As shown in Figure 1, total symptom scores were significantly elevated by day 2 postinoculation compared with preinoculation (15.3:±:3.5 vs 1.:±:.5, p<.1 Bonferroni) and peaked on day 3 (15.4:±:3.2, p:::;o.ol Bonferroni). Thereafter, there was a gradual decline in the symptom scores to baseline by day 8. Subjects were allowed the use of acetaminophen for symptomatic relief. This was used by nine patients. Only two patients (No. 5 and No. 9) required the use of oral pseudoephedrine on days 6 and 7 of the study. Patient 5 was started on a regimen of azithromycin for otitis media on day 7 of the study. Patient 8 required diphenhydramine and lorazepam on day 6, 7 and 8, respectively, for sedation needed for a previously undisclosed psychiatric illness. NO Studies NO was detectable in the exhaled oral and nasal breath of all study subjects. At baseline, nasal NO levels were fivefold (mean:±:se ppb; right, 28.4:±:3.7; left, 27.7:±:4.6) higher than oral NO levels (mouth NO, 5.8:±:.3 ppb, p<o.ool vs either nasal NO value) (Fig 2). Oral NO levels declined slightly over the first 4 days after inoculation (Fig 3) and increased significantly at day 8 compared with baseline values (analysis of variance, p:::;.1; Bonferroni 454 z p <.1 p <.1 s 12 n.s. c&, qs> o o Mouth Nose - Rt Nose - Lt FIGURE 2. Mouth vs nasal NO levels on day prior to inoculation \vith virus. Comparison is made by Student paired t test. p<.1). There was a slight decrease in nasal NO on days 2 through 4 and return to just above baseline on day 8 but these changes did not reach significance (Table 2). Correlations Among NO, Symptoms, and Viral Replication The peak of the total symptom score did not correlate temporally with the peak exhaled NO (Figs 1 and 3). Consequently, we further subdivided patient symptom scores into upper respiratory symptoms (nasal stuffiness, runny nose, and sneezing) and lower respiratory symptoms (cough and breathing difficulties) to see if any association existed. The peak for the upper respiratory symptoms was on day 3 (5.9:±:1.1) with a gradual decline to baseline. Lower respiratory symptoms also peaked on day 3 but at a much lower level (.5:±:.2). Like the total respira- 2,-.., _, 1 z..c: ceo "5 5 O<DO ~ * 21 Days Post Inoculation FIGURE 3. Mouth NO levels from day through day 21 postinoculation, p< O.Ol by Bonferroni.

4 Table 2-Mean Expired Mixed NO Day No. Right Nares Left Nares 28.4::+: ::+: ::+: ::+: ::+: ::+: ::+: ::+: ::+: ::+: ::+: ::+: ::+: ::+: 1.9 tory symptoms score, upper and lower symptom scores were not associated temporally with the peak in expired NO. Similarly, the magnitude of the peak in nasal or throat viral titers (days 2 to 3) did not correlate with the later rise in oral NO (day 8). When the peak nasal wash viral titer for each individual was analyzed with respect to the change from baseline in oral NO, no significant correlation was found. DISCUSSION We have observed that infection of healthy individuals with influenza virus can elevate oral mixed expired NO, and that this elevation, at day 8, is not associated temporally with patient symptoms or peak virus recovery. Previous work evaluated asthmatic patients presenting with symptoms of an "upper respiratory tract infection" and did not identify which viruses were responsible for the infection. In addition, the authors did not follow exhaled levels of NO prior to infection through recovery.l 2 Thus, they were not able to correlate a specific viral infection with the changes in exhaled NO levels. In the present work, we were able to document that influenza A infection can increase exhaled mixed oral N levels at 8 days postinfection and this increase was not associated with patient symptoms, implying that NO may not be directly responsible for the respiratory tract symptoms of infection with influenza A. This finding of an increase in mixed expired oral NO must be interpreted with caution. Mean expired oral NO was not measured on days 5, 6, 7, and at only one time point after day 8; thus, a gradual rise and fall in N was not observed. The possibility that the elevation of NO observed was an artifact of the collection procedure seems unlikely. The NO analyzer was calibrated daily with standard gases ( and 1 ppm) and a mixed expired oral NO obtained from a control volunteer (healthy nonasthmatic who was not exposed to influenza A) on each day that the NO analyzer was used revealed no elevation of mixed expired oral NO. Given that the volunteers in this study had no important respiratory tract or systemic complaints on day 8, it seems unlikely that they suddenly developed any of the illnesses that are known to elevate oral NO. The increase in expired oral NO was not associated vvith a concomitant increase in expired nasal NO, suggesting that nasal NO does not make significant contributions to exhaled oral NO. This is consistent with the observations of Massaro and colleagues 1 who have shown that the elevation of N seen in asthmatics and normal subjects is from the lower airways. Furthermore, recent guidelines from the European Respiratory Society suggest that when measuring oral exhaled NO, a back pressure of 5 to 2 em H 2 measured at the mouth should be generated to isolate the upper respiratory tract from the lower respiratory tract. 18 The collection of NO into a Mylar bag using an FVC maneuver fulfills these criteria. Finally, rhinopharyngoscopy demonstrates that the nasopharynx closes during FVC exhalation (data not shown). Thus, we believe that collecting oral exhaled N through the use of an FVC maneuver through a plastic tube into a polyester film balloon represents gas obtained from the lower airways and that elevation of mixed expired oral NO seen herein is not the result of contamination from the upper airways. The present report demonstrates an increase in exhaled NO gas occurring 8 days after experimental inoculation of influenza virus in normal volunteers. These patients develop marked upper respiratory tract symptoms that coincide with peak viral cultures (ie, 2 to 4 days) but not with elevation of NO. Our observation of increased exhaled N after viral infection in the absence of lower respiratory tract symptoms suggests that, in normal individuals, elevated N levels may not be responsible for lower respiratory tract symptoms. Whether these events relate to exacerbation of asthma during naturally occurring virus infection is not known. ACKNOWLEDGMENTS: The authors wish to thank Drs. Ben Gaston and Lisa Wheatley for advice on the manuscript. REFERENCES 1 Aston B, Drazen JM, Loscalzo J, eta!. The biology of nitrogen oxides in the airways. Am J Respir Crit Care Med 1994; 149: Gustfsson LE, Leone AM, Persson MG, et a!. Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. BBRC 1991; 181: Persson MG, Zetterstrom, Agrenius V, eta!. Single-breath nitirc oxide measurements in asthmatic patients and smokers. Lancet 1994; 343: Alving K, Weitzberg E, Lundberg JM. Increased amount of nitric oxide in exhaled air of asthmatics. Eur Respir J 1993; 6: Massaro AF, Gaston B, Kita D, et a!. Expired nitric oxide levels during treatment of acute asthma. Am J Respir Crit Care Med 1995; 152:8-3 CHEST /1 14 /2 I AUGUST,

5 6 Kharitonov SA, Yates D, Robbins RA, et a!. Increased nitric oxide in exhaled air of asthmatic patients. Lancet 1994; 343: Yates DH, Kharitonov SA, Robbins RA, eta!. Effect of a nitric oxide synthase inhibitor and a glucocorticosteroid on exhaled nitric oxide. Am J Respir Crit Care Med 1995; 152: Kharitonov SA, Yates DH, Barnes PJ. Inhaled glucocorticoids decrease nitric oxide in exhaled air of asthmatic patients. Am J Respir Crit Care Med 1996; 153: Martin U, B1yden K, Devoy M, et a!. Increased levels of exhaled nitric oxide during nasal and oral breathing in subjects with seasonal rhinitis. J Allergy Clin Immunul 1996; 97: Massaro AF, Mehta S, Lilly CM, et a!. Elevated nitric oxide concentrations in the isolated lower ainvay gas of asthmatic subjects. Am J Respir Crit Care Med 1996; 153: Robbins RA, Floreani AA, Von Essen SG, et al. Measurement of exhaled nitric oxide by three different techniques. Am J Respir Crit Care Med 1996; 153: Kharitonov SA, Yates D, Barnes PJ. Increased nitric oxide in the exhaled air of normal human subjects with upper respiratory tract infections. Eur Respir J 1995; 8: Nain M, Hinder F, Gong J, et al. Tumor necrosis factor-a production of influenza A virus-infected macrophages and potentiating effect of lipopolysaccharide. J Immunol 199; 145: Tomoda T, Morita H, Kurashige T, et a!. Prevention of influenza by the intranasal administration of cold-recombinant, live-attenuated influenza virus vaccine: importance of interferon-'y producation and local IgA response. Vaccine 1995; 13: Robe1ts N, Prill A, Mann T. Interleukin l and interleukin 1 inhibitor production by human macrophages exposed to influenzas virus or respirat1y syncytial virus. J Exp Med 1986; 163:5ll Peper R, Campen H. Tumor necrosis factor as a mediator of inflammation in influenza A viral pneumonia. Microbial Pathogenesis 1995; 19: Hayden FG, Fritz RS, Lobo MC, et a!. Local and systemic cytokine responses during experimental human influenza A virus infection: relation to symptom formation and host defense. J Clin Invest 1998; 11: Kharitonov S, Alving K, Barnes PJ. Exhaled and nasal nitric oxide measurements recommendations: the European Respiratory Society Task Force. Eur Respir J 1997; 1: