Bronchial Hyperresponsiveness in Normal Subjects during Attenuated Influenza Virus Infection 1- 3

L. A. LAITINEN, R. B. ELKIN,4 D. W. EMPEY,5 L. JACOBS, J. MILLS, and J. A. NADEL

Introduction

It has been suggested that abnormalities of airway epithelium cause bronchial hyperresponsiveness (1-3). In support of this hypothesis, Empey and associates (4) demonstrated transient bronchial hyperresponsiveness in otherwise normal subjects with symptomatic respiratory tract infections and presumed airway epithelial damage (5). However, the etiology and exact time of onset of the infections was not determined, and the study was dependent upon the occurrence of spontaneous infections in the community. To overcome these difficulties in the present study, we produced bronchial hyperresponsiveness in healthy subjects by administration of live attenuated influenza virus. Methods We studied 14 healthy male subjects 21 to 36 yr of age, who had hemagglutinationinhibition antibody titers of 1:8or less to homologous influenza A virus. All subjects were fully informed volunteers who signed consent forms approved by the committee on Human Research of the University of California, San Francisco, before participating in the study. None of the subjects had a history of asthma, hay fever, allergic dermatitis, or conjunctivitis. The subjects had no symptoms of cardiopulmonary disease and had not had symptomatic respiratory tract infections during the 2 months preceding the study. 1\vo of the subjects smoked 10to 20 cigarettes per day. The general physical examination of the subjects was normal, and they had normal lung function. On each day of the study, we graded and recorded each subject's complaints using a standardized respiratory symptom questionnaire and performed a general physical examination (6). The subject then inhaled 10 tidal-breath aerosols of 0.4 and 1.6070 solutions of histamine diphosphate in normal saline, prepared daily and buffered to pH 7.0 with sodium bicarbonate. Normal saline was used as a control aerosol. We delivered the solutions from a DeVilbiss no. 40 glass nebulizer (DeVilbiss Co., Somerset, PA) with a constant flow of 10L/min of compressed air, producing a volume median droplet diameter 358

SUMMARY Fourteen healthy male .ubJects with hemagglutination-Inhibition antibody tltera of 1:8 or Ie.. to homologou.lnfluenza A vlru. were .tudled. Six .ubJects received live, attenuated Influenza vlru. by na881 drop. and by aerosol. Although Infection occurred In the. . .Ix .ubJects, with the development of 4-fold or greater Increa... In hemagglutination-Inhibition antibody tltera, they remained asymptomatic. Eight .ubJects received placebo via the .ame route, and did not develop symptom. and .howed no Increa.. In antibody titer. Prior to admlnl.tration of vlru. or placebo, hl.tamlne dlpho.phate aeroaollncreaaed airway re.l.tance only .lIghtly, and there we. no difference between the vlru. and placebo groups. 'tWo days after Inoculation, bronchomotor re.pon... In the placebo group were unchanged (p > 0.05), but In the vlru.-Infected group, bronchomotor re.pon... were .Ignlflcantly greater than In the prelntected .tate (p < 0.01). Isoproterenol hydrochloride reversed and prevented the Increa.. In airway re.l.tance after hl.tamlne, .ugge.tlng that caused by .mooth muscle contraction. Our finding. Indicate that tranthe bronchocon.trlctlon .Ient, asymptomatic re.plratory vlru. Infection augment. airway .mooth muscle re.pon....

we.

/.aM REV RESPIR DIS 1991; 143:358-381

of approximately 4.5 J.1m (7). The output of the nebulizer was 0.5 ml/min. Subjects were unaware of the sequence of the aerosols and were told that the inhalations might make them feel better or worse or have no effect. Airway resistance and thoracic gas volume were measured using a 900L constant-volume whole-body plethysmograph (8). Recordings were made using a rapid-writing photographic recorder (DR-12; Electronics for Medicine, White Plains, NY). We measured airway resistance every 30 s for 5 min prior to the inhalations of aerosols, and we reported the mean of these measurements as the baseline value. After the inhalation of histamine, we repeated the measurements every 30 s for 5 min, or longer if airway resistance remained elevated above the baseline value. The mean of the measurements during the 5 min after inhalation of histamine is reported as the response to the aerosol. To determine whether the increase in airway resistance produced by histamine could be prevented, we administered isoproterenol hydrochloride aerosol (0.5070, two breaths) to the subjects. We then obtained new baseline measurements of airway resistance, and 10 min after inhalation of isoproterenol we administered histamine aerosol and measured airway resistance as before. Whenever we attempted to prevent the responses to histamine, further studies were performed later the same day or the next day to determine whether hyperresponsiveness to histamine was still present. To determine whether the bronchoconstriction induced by histamine could be reversed, we gave isoproterenol aerosol to subjects 4 min after inhalation of histamine. We

then measured airway resistance as before and used the mean of the seven values obtained after either sequence to determine the effect of isoproterenol aerosol on the histamineinduced bronchoconstriction. The subjects were assigned randomly to groups that received either placebo or live virus as nasal drops and aerosol. Eight subjects (controls) including the two who smoked, received the placebo, uninfected avian allantoic fluid. The remaining six subjects received attenuated inhibitor-resistant strains of influenza virus grown in the allantoic cavity of hen's eggs, supplied by Smith, Kline, and French (9-11). These subjects receivedeither influenza A (H3N2; Alice strain) and B (a strain of B Hongkong 5 72) together, or they receivedinfluenza A (H3N2; strain RJT 4025) alone, 107 •5 50070 infectious doses. Responses to both

(Received in original form December 19, 1989 and in revised form August 17, 1990) 1 From the Cardiovascular Research and Departments of Medicine and Physiology, University of California, San Francisco, California. 2 Supported in part by Specialized Center for Research in Pulmonary Disease Grant HL-14201 from the National Heart and Lung Institute. 3 Correspondence and requests for reprints should be addressed to Lauri A. Laitinen, M.D., AB Draco, Explorative Clinical Research, P.O.Box 34, S-221 00 Lund, Sweden. 4 Recipient of fellowships from the San Francisco Lung Association and the California Lung Association. S Recipient of International Research Fellowship FOSTW2129 from the U. S. Public Health Service.

359

HYPERRESPONSIVENESS AND INFLUENZA VIRUS INOCUL ATIO N

virus inoculations were similar, and the data were pooled. We measured convalescent hemagglutination-inhibition antibody titers to the homologous influenza A virus at 16to 20 days after administration of virus or placebo, using standard technique (12). We compared acute and convalescent titers directly in a single test and considered 4-fold or greater increases significant (12). Virus recovery was attempted by inoculating throat gargles into rhesus monkey cell culture, and testing for hemadsorption after 14 days of culture (6, 12). Each subject's response to histamine was established before administration of virus or placebo and at 2, 4, and 9 days afterwards. Statistical significance (p 0.05) (figure 3). Discussion

Our study demonstrates a significant increase in bronchial responsiveness to inhaled histamine aerosol in five of six subjects after administration of live, attenuated influenza virus; none of the subjects who received placebo developed hyperresponsiveness. The finding that increased bronchial responsiveness was associated with 4-fold or greater increases in homologous hemagglutination-inhibition antibody titers in five of six subjects strongly suggests that hyperrespon-

Airway Resistance Fig. 2. Effect of histam ine aerosol on airway resistance in six subjects studied 2 days after administration of virus and who subsequently developed 4-fold or greater increases in hemagglutination-inhibition antibody titer to influenza virus. Each ciosed symbol rapresents a single measurement of airway resistance prior to administration of virus. Baseline resistances were measured over a 5-min period but are pooled for convenience. Each open symbol represents a single measurament of airway resistance 2 days after administrat ion of virus . Hatched bars indicate time at which histamine diphosphate aerosol (1.6%) was administered.

(em HzO/LPS)

Subject

Sub/eel

Sub/eel

I

2

.3

8

4

o

'L

- - - - - "_ - " - - - - - '

4[ ' o

~

I

-3

~ j 0

~

4

6

~ ~ ~

[

~

I

I

I

3

6

-3

T im e (min)

~

§ 0

I

I

3

6

LAITINEN, ELKIN, EMPEY, JACOBS, MILLS, AND NADEL

360 ISOPROTERENOL

6

4

2

Airway Resistance After Histam ine

6

o

(ern H20 / LPS) 4

Airway Resistance Before Histam ine (ern H20 / LPS)

Fig. 3. Etfect of isoproterenol hydrochloride aerosol (0.5%, two breaths) on modifying responses to lnhatation ofhistamine diphosphate aerosol (1.6%,10breaths) in four subjectsafter administration of virus. The line ofidentity is drawn; points abovethe lineindicate bronchoconstriction, pointsbelow theline indicate bronchedilation, and points on the lineshowno change inairway resistance. Each pointis the mean of seven measurements of airway resistance before and after histamine inone subject. Eachpanelshowsthe responsetolnhalation ofhistamine aerosol before (open circles) andafter (closedcircles)isoproterenol intervention. Isoproterenol reversed (upperpanel) and prevented (lowerpenel)the increaseinairway resistanceafter inhalation of histamine aerosol.

siveness was related to infection. However, the hyperresponsiveness was not associated with respiratory signs or symptoms, consistent with previous reports that infection with this live, attenuated influenza virus is usually asymptomatic (9-11). We were unable to recover the virus from infected subjects, again consistent with previous studies with this attenuated virus that have reported a virus recovery rate of 5 to 15070 (9-11). However, the period of bronchial hyperresponsiveness coincided with the period of successful virus isolation reported by Hall and colleagues (18) after administration of attenuated influenza virus. The importance of infection in causing bronchial hyperresponsiveness is also suggested by failure to demonstrate increased responses to methacholine in normal subjects after subcutaneous administration of inactivated polyvalent influenza vaccine (19). In that study, the increased bronchial responsiveness after vaccina-

tion of asthmatic subjects is unexplained, but it may be related to systemic effects resulting from administration of the vaccine. We can exclude several mechanisms previously suggested as explanations for bronchial hyperresponsiveness in patients with asthma and chronic bronchitis because they are not applicable to our normal subjects: the presence of airway narrowing prior to inhalation of histamine aerosol (20) cannot explain our findings because baseline airflow resistance did not change after administration of virus. Others have also failed to demonstrate evidence of airway obstruction after intranasal administration of attenuated influenza virus (18). Similarly, our findings in normal subjects cannot be explained by bronchial smooth muscle hypertrophy and hyperplasia (21-23) or by the use of drugs that might complicate interpretation of results. Small doses of isoproterenol hydrochloride aerosol prevented and reversed the increases in airway resistance after histamine, suggesting that these increases were due to contraction of airway smooth muscle and were unlikely to be caused by partial beta-adrenergic receptor blockade (24). None of the five subjects who developed bronchial hyperresponsiveness had histories suggestive of atopy; each of the five hyperresponsive subjects available for further studies had normal serum IgE levels, and four of these five subjects had completely negative skin tests. It is also unlikely that administration of virus initiated antigenantibody reactions that were responsible for the development of bronchial hyperresponsiveness because our subjects had low initial levels of serum hemagglutination-inhibition antibodies to attenuated influenza virus and because the known serum and secretory antibody responses to respiratory virus infection usually occur too late to coincide with the period of transient bronchial hyperresponsiveness (6, 25, 26). Viral upper respiratory tract infections cause transient and reversible damage to airway epithelium (27, 28). The magnitude and duration of bronchial hyperresponsiveness caused by spontaneous, symptomatic viral infections (4) exceeded that caused by asymptomatic infection with an attenuated virus seen in this study. We suggest that this is due to a greater degree of inflammation and airway epithelial damage in infections caused by wild-type viruses. This is supported by the observation that all of our subjects were asymptomatic, whereas the

subjects studied with spontaneous viral infections were all symptomatic. Epithelial injury might also permit greater access of airway smooth muscle to inhaled histamine, and this could be a cause of bronchial hyperresponsiveness. It is often assumed that the occurrence of IgE-mediated antigen-antibody reactions within the respiratory tract, with subsequent release of mediators (such as histamine), is sufficient to cause episodes of bronchospasm. However, the evidence is that mediators produce little change in airway resistance in the absence of bronchial hyperresponsiveness. Thus, the occurrence of bronchospastic disorders may require the presence of two independent abnormalities: bronchial hyperresponsiveness, perhaps as a result of airway inflammation, and a bronchoconstrictor stimulus (e.g.; histamine). Furthermore, the well-recognized deterioration of asthmatic and bronchitic patients when they suffer viral respiratory tract infections (29) may be due to inflammation and the subsequent increase in airway responsiveness. We conclude that asymptomatic viral infections cause augmented airway smooth muscle responsiveness in otherwise healthy subjects.

Acknowlegment The writers thank Dr. J. Schieble for help with determination of antibody titers and Drs. O. Frick, R. Freinkel, and S. Tolber for performance of skin tests. References 1. Nadel JA. Structure-function relationships in the airways:bronchoconstriction mediated via vagus nerves or bronchial arteries; peripheral lung constriction mediated via pulmonary arteries.Med Thorac 1965; 22:231-42. 2. Nadel JA. Neurophysiologic aspects of asthma. In: Austen KF, Lichtenstein LM, OOs. Asthma: physiology, immunopharmacology, and treatment. First International Symposium. New York: Academic Press, 1973:29-37. 3. Laitinen LA, Heino M, Laitinen A, Kava T, Haahtela T. Damage of the airwayepithelium and bronchial reactivity in patients with asthma. Am Rev Respir Dis 1985; 131:599-606. 4. EmpeyDW, Laitinen LA, Jacobs L, Gold WM, Nadel JA. Mechanisms of bronchial hyperractivity in normal subjects following upper respiratory tract infection. Am Rev RespirDis 1976; 113:131-9. 5. Hers JFPH. Disturbances of the ciliated epithelium due to influenza virus. Am RevRespir Dis 1966; 93:162-71. 6. Van Kirk JE, Mills JV, Chanock RM. Evaluationoflow temperature grown influenza A2/Hong Kong virus in volunteers. Proc Soc Exp Bioi Med 1971; 136:34-41. 7. MercerIT. Production and characterization of aerosols. Arch Intern Moo 1973; 131:39-50. 8. DuBoisAB, Botelho SY,Comroe JH Jr. A new method of measuringairwayresistance in man using

361

HYPERRESPONSIVENESS AND INFWENZA VIRUS

a body plethysmograph: values in normal subjects and in patients with respiratory disease. J Clin Invest 1956; 35:327-35. 9. Rytel MV, Jackson LS, Ferstenfeld JE, Rosenkranz MA. New live attenuated influenza A/England/42/72 (H3N2) vaccine (Alice): reactogenicity, immunogenicity, and protection efficacy. J Infect Dis 1975; 132:652-9. 10. Minor TE, Dick EC, Dick CR, Inhorn SL. Attenuated influenza A vaccine (Alice) in an adult population: vaccine-related illness, serum and nasal antibody production, and intrafamily transmission. J Clin Microbiol 1975; 2:403-9. 11. Spencer MJ, Cherry JD, Powell KD, Sumaya CV. A clinical trial with Alice/R-75 strain, live attenuated serum inhibitor-resistant intranasal bivalent influenza AlB vaccine. Med Microbiol Immunol (Bert) 1979; 167:1-9. 12. Robinson RQ, Dowdle WR. Influenza viruses. In: Lennette EH, Schmidt NJ, eds. Diagnostic procedures for viral and rickettsia infections. American Public Health Association, 1969; 414-33. 13. Snedecor OW, Cochran Wo. Statistical methods. Ames, Iowa: Iowa State University Press, 1967. 14. Wide L, Porath J. Radioimmunoassay of proteins with the use of Sephadex-coupled antibod-

ies. Biochim Biophys Acta 1966; 130:257-60. 15. Johansson SOO, Bennich H, Wide L. A new class of immunoglobulin in human serum. Immunology 1968; 14:265-72. 16. Johansson SOO. Serum IgND levelsin healthy children and adults. Int Arch AllergyAppl Immunol 1968; 34:1-8. 17. Sheldon JM, LovellRO, Mathews KP. A manual of clinical allergy. Philadelphia: W B Saunders, 1967. 18. Hall WJ, Douglas RO Jr, zaky DA, Hyde RW, Richman D, Murphy BR. Attenuated influenza virus in normal adults: role of pulmonary function studies in vaccine trials. J Infect Dis 1976; 133: 145-52. 19. Quellette JJ, Reed CEoIncreased response of asthmatic subjects to methacholine after influenza vaccine. J Allergy 1965; 36:558-63. 20. Benson MK. Bronchial hyperreactivity. Chest 1975; 69:227-39. 21. Dunnill MS, Massarella OR, Anderson JA. Comparison of the quantitative anatomy of the bronchi in normal subjects, in status asthmaticus, in chronic bronchitis, and in emphysema. Thorax 1969; 24:176-9. 22. Hossain S. Quantitative measurement of bron-

chial muscle in men with asthma. Am Rev Respir Dis 1973; 107:99-109. 23. Hossain S, Heard BE. Hyperplasia of bronchial muscle in chronic bronchitis. J Patho11970; 101:171-84. 24. Szentivanyi A. The beta-adrenergic theory of the atopic abnormality in bronchial asthma. J Allergy 1968; 42:203-32. 25. Cate TR, Rossen RD, Douglas RO Jr, Butler WT, Couch RB. The role of nasal secretion and serum antibody in the rhinovirus common cold. Am J Epidemiol 1966; 84:352-63. 26. Rossen RD, Butler WT, Waldman RH, et al. The proteins in nasal secretion. II. A longitudinal study of IgA and neutralizing antibody levels in nasal washing from men infected with influenza virus. JAMA 1970; 211:1157-61. 27. Walsh JJ, Diethlein LF, Low FN, Burch OB, Mogabgab WJ. Bronchotracheal response in human influenza. Arch Intern Med 1961; 108:376-82. 28. Hoorn B, Tyrell DAJ. On the growth of certain "newer" respiratory viruses in organ cultures. Br J Exp Pathol 1965; 46:109-18. 29. Lambert HP, Stem H. Infective factors in exacerbations of bronchitis and asthma. Br Med J 1972; 3:323-6.