Effects of experimental rhinovirus 16 infection on airways and leukocyte function in normal subjects

ects of experimental rhinovir ection on airways and leuko ction in normal subjects Bush, M.D., William Busse, M.D., Dennis Flaherty, Ph.D., rshauer, Ph.D., Elliot C. Dick, Ph.D., and Charles E. Reed, Madison,

Wis .

we infected 7 normal volunteers with rhinovirus 16. In general, the symptoms and alterations in airways were minimal although all subjects had upper respiratory symptoms. Three subjects developed in addition lower respiratory and systemic symptoms accompanied by either an increase in the volume of isojow or a positive methacholine response. Furthermore, these three had a decrease in beta adrenergic and Hz histamine receptor responses of their granulocytes. Since the peripheral airway obstruction and decreased beta adrenergic and Hz histamine responses occurred together, these two phenomenon may have a common cause. All seven subjects had a decrease in number of circulating E rosette-forming lymphocytes, and in 6 of 7, there was a decrease in the antibody-dependent cellular cytotoxicity capacity of mononuclear cell preparations. It is not clear whether these changes reflect redistribution of mononuclear cells or alteration of their function.

Respiratory virus infections (including rhinovirus) often provoke episodes of wheezing in persons with asthma.lm3 Changes in airway function during the course of viral respiratory infections may not be limited to persons with asthma. Increased frequency dependence of compliance,4 decreased diffusion capacity, increased closing volume,6 and decreased maximal flow rates with helium-oxygen mixtures6 have been found in normal persons with viral respiratory infections. Normal individuals with such infections may also demonstrate increased bronchial reactivity to aerosols of methacholine7 and histamine.8 The mechanisms responsible for these in vivo observations are not well understood. Cells from subjects with asthma show altered pba~acologic responses in vitro.ga lo Using inhibition of lysosomal enzyme release from granulocytes (PMNs) as a model of beta adrenergic response, ussel’ has shown a diminished response to isoproterenol in asthmatics; and during naturally acquired

Supported by Allergic Disease Center Grant No. Z-P15-AI-10404, Specialized Center of Research Grant No. l-P17-HL-15389, and Autonomic Grant No. 2-ROl-AI-08106. Received for publication June 1, 1977. Accepted for publication Oct. 26, 1977. Reprint requests to: Robert K. Bush, M.D., 504 N. Walnut St., Madison, Wis. 53706. vd.

61,

NO.

2, PP. 80-87

respiratory infections provoking attacks of asthma, the PMN response to isoproterenol was further reduced. Employing a similar model, man12 have demonstrated a decreas tamine response of PMNs of asthmatics. The effect of viral infection on the PMN responses in notmal persons is not known. Asthma may also be associated with altered immunologic responses in vitro. Flaherty and associates13have reported a diminished ~tibody-de~er~d~nt cellular cytotoxicity capacity (ADCC) of mononuclear cells from patients whose attacks of asthma were triggered by respiratory infections. However, the role of diminished ADCC capacity in the elicitation of virus-provoked asthma is not known. Viral infections do produce lymphopenia, and several studies have shown alterations in the normal distribution of immunocompetent cells.14-16 Our present study of 7 normal subjects deliberately infected with rhinovirus 16 (RV16) suggests that such infections may result in airway changes associated with decreased in vitro responsiveness of granulocytes to isoproterenol and histamine. METHODS AND MATERIALS Patient selection Seven healthy adults, 2 females and 5 males, 20 to 28 yr

of age, volunteered for the study. All were nonsmokerswho 0091-6749/78/0261-0080$00.8010

0

1978

The

6. a/. ~0s~~

Co.

VOLdME 61 NUMBER 2

ERects

did not have asthma, allergic rhinitis, or atopic eczema. None had any significant respiratory disease nor a recent viral illness. None were taking medications. All had an FEVi within 90% of predicted normal values17 and had no detectable antibody to RV16.

The challenge virus was prepared as described previ0us1y.‘~ Briefly, the inoculum was a safety tested, secondpassage harvest of RV16 from human embryonic fibroblast cell cultures (WI-38). This strain was isolated from a person with a naturally acquired common cold. Each person was inoculated intranasally with 5.6 X 103-5.6 x lo5 TCIDjos of RV16 suspended in 1 ml of Hanks balanced salt solution (HBSS) (Table I). The inoculum was administered with the subject in the supine position, with the head extended, during and for 3 min after inoculation. Each subject was instructed not to blow the nose for at least 30 min after inoculation.

I wa~he§ Nasal washings were obtained immediately prior to inoculation and daily for 7 days thereafter (Table I) using methods previously described.la Five ml of HBSS supplemented with 0.5% gelatin was instilled into each nostril while the subject was in a sitting position with the head in hyperextension. The solution was expelled through the nares into a sterile receptacle.

irus isolation Preinoculation nasopharyngeal specimens were inoculated into four cell culture systems: WI-38,18 fetal tonsil,lg primary rhesus monkey kidney,‘* and HEp-2.‘* Specimens obtained after inoculation were placed only in WI-38 and fetal tonsil cells. RV16 isolates were identified by neutralization with specific antisera. Recovery of the challenge virus on one or more occasions after inoculation was considered as evidence of an RV16 infection.

titers Ten milliliters of serum were obtained at baseline, 3, 7, 14, 21, and 28 days. Neutralizing antibody titer to RV16 was carried out by methods described elsewhereis in WI-38 cells with lo-100 TCIDS,,s as the challenge dose. A fourfold or greater rise in serum neutralizing antibody titer one month after inoculation was presumptive evidence for an infection with RV16.

symptoms A physician evaluated each subject on day 0,3,7, and 21 for the presence of fever, rhinitis, pharyngitis, and pulmonary findings. Each person filled out a daily symptom score sheet with the following scale: 0 = not present, 1 = mild, 2 = moderate, 3 = severe. Signs and symptoms were categorized into three groups: (1) upper respiratory (rhinorrhea, nasal obstruction, sneezing, and pharyngitis); (2) lower respiratory (cough and chest tightness); (3) systemic (fever, chills, malaise, and headache).

of rhinovirus

TABLE

Subject

1 2 3 4 5 6 7

on airways

Challenge dose (TCID,.,)

Pulmonary

ieukocyte

function

studies

I. Virus

5.6 5.6 5.6 5.6 5.6 5.6 5.6

and

x x x x x x x

Days virus recovered

lo3 lo3 lo3 lo3 lo3 1Oj 1Oj

1, 2, 4, 6, 1, 2, 4, 6, 2, 6, 2, 3, 4, 6, None 1, 4,

function

tests

7 4 7 7 7 6

~1:2.8 ZZ1:2.8 ~1:2.8 rl:2.8 ~1:2.8 <1:2.8 <1:2.8

1:128 lZ90.5 ~115.6 %1:.5.6 ~1:5.6 1: 128 I:256

1. Forced expiratory volume in one second (FEV,), maximal midexpiratory flow (FEF25%-75%), and forced vital capacity (FVC) were recorded on a Collins spirometer. All values were corrected to standard conditions (BTPS). Three tracings were obtained each time, and the one with the largest vital capacity was used for measurements. 2. Methacholine challenges were performed by the method of Parker, Bilbo, and Reed. i A fall in FEV, of 15% or greater, 5 min after inhalation of methacholine was considered a positive test. 3. Maximum expiratory flow-volume curves while breathing an 80% helium-20% oxygen mixture (He-Q) and room air were obtained and analyzed as described by Storms, Dick, and Busse.20 A volume body plethysmograph was used for flow-volume determinations. A Med-Science wedge spirometer was attached to the plethysmograph via a large-bore port. Flow was measured at the mouth with a Fleisch No. 3 pneumotachometer, open to room air through a port in the plethysmograph. Flow and volume were recorded on an Esterline Angus XY recorder. The pneumotachometer was standardized for flow with the two gas mixtures by measuring the volume delivered with a Tissot gasometer over a given period of time at a specified flow rate. Corrections in the recorded flow rate were made so that the delivered volumes were equal. Each subject performed flow volume curves while breathing room air; tracings were recorded until three curves with vital capacities within 10% of each other were obtained. The patient then inhaled three deep breaths of the He-O2 mixture and breathed at tidal volume for 2 minutes. Three more flow-volume curves (within 10% of each other) were obtained. Flow rates while breathing room air and the He-O* mixture were calculated after 50% (Vmax& and 75% (\jmax& of the vital capacity had been expelled. Evaluation of small airways function, volume of isoflow (VisoV), was made using a comparison of air Bow volume curves with flow volume curves done with the He-Q mixture. The air flow curve was superimposed visually upon the He-O2 curve. The vital capacities of the curves selected for matching were identical in all but Subject 5 in whom the vital capacity on He-O2 was larger than on air. The curves

Bush et a!.

$1. Pulmonary

T

function

tests

Spirometry ubject

% pred

Day

I

3

4

5

6

7

% pred

108 102 103 104 110 103 111 104 100 94 99 112 100 106 94 91 97 107 108 110 97 102 110 99

0

3 7 21 0 3 7 2i 0 3 7 0 3 7 0 3 7 0 3 7 0 3 7 21

2

FEV,

FVC

111

104 106 107 112 109 116 112 99 96 102 104 102 96 96 96 101 96 98 97 115 114 118 113

% pred

FEF25y0.-75y0

92 78 81 78 89 82 91 77 94 83 86 107 94 95 80 75 78 136 122 115 64 68 75 54

X d~~re~5e

FEV,

5 1 3 2 5 0 7 0 7 2 1 3 0 0 4 1 5 2 0 0 10 25 24 21

*Measured from residual volume. were aligned at the beginning (total lung capacity) and at the end (residual volume) of expiration. The point at which the flows become identical is the VisoV. Normal subjects (N = 17) in our laboratory have a mean & SD VisoV of 7 t 9.5. @II

aration

Anticoagulated blood (0.5 ml 2.7% EDTA/9.5 ml blood) was obtained for leukocyte studies on days 0, 3, 7, and 21 and was separated by Ficoll-Hypaque density centrifugation as described by Boyum.21 After centrifugation (500 g X 40 min at 20” C), mononuclear cells were recovered from the Ficoll interface, washed in medium 199 (Gibco, Grand Island, N. Y.), and resuspended to a final concentration of 7 x 106 cells/ml. The remaining Ficoll-Hypaque was discarded. The granulocytes isolated from the cell pellet was resuspended in HBSS at a final concentration of 3 x lo6 WIN/ml (98% PMNs).

al enzyme

determination

Replicate l-ml samples of PMN suspensions (3 X lOs/ml) were placed into 10 X 75 mm plastic test tubes and preincubated with cytochalasin B (5 pg/ml x 5 min) at 37”

C. Cytochalasin B (CB) allows for selective enzyme release independently of particle ingestionz2 In testing the granulocyte response to agonists, isoproterenol, and histamine, the PMNs were preincubated with theophylline (5 X 1O-5 M for 15 min) before incubating with isoproterenol (X 15 min) or histamine (X 15 mm). This concentration of theophylline by itself did not change enzyme release. Sodium metabisulfite (0.01% final concentration) was added to the freshly prepared isoproterenol to reduce oxidation. The methods employed in zymosan-induced lysosomal enzyme release were those described previously.23 Briefly zymosan was boiled in saline (10 mg/ml X 10 mm, washed X 2, and suspended in HBSS (10 mgiml). Following incubation with CB and agonists, autologous serum 0. I ml and 0.1 ml of the freshly prepared zymosan were added. Following 30 min incubation in a 37” C shaking water bath (120 cycles/min), the reaction was stopped by centrifugation (750 g X 10 min at 4” C). The cell-free supematant was removed for enzyme determination. Total PMN enzyme activity was obtained following cell lysis (0.2% Triton X-100). The lysosomal enzyme beta glucuronidase (BC) was determined after 18 hr incubation at 37” C with phenolphtbalein glucuronidate as substrate.24 Percent inhibition of en-

Effects

Helium-oxygen max50

air

Vmaxeo He-02 (Llsec)

(LhC)

3.0 3.1 3.0 2.9 4.0 4.8 3.8 4.2 3.4 4.0 4.2 4.9 3.8 4.1 4.3 3.9 4.0 6.0 8.8 5.7 3.0 3.0 3.3 1.6

to untreated cells. To determine cell viability, the cytoplasmic enzyme lactate dehydrogenase was also measured in the supematant . 25

counts

and differentials

Total white blood cell differential on days 0, 3, 7, and 21.

counts were performed

e determinations Absolute lymphocyte counts, E rosette (T lymphocyte), EAC mouse rosettes (B lymphocytes), and Ig-EA rosettes were performed as described elsewhere.13

Antibody-dependent

cellular

air

1.5 1.6 1.5 1.4 1.8 2.5 1.9 1.8 2.0 2.1 2.8 3.3 2.1 2.3 2.0 1.9 1.9 3.4 5.5 3.4 1.3 1.2 0.8 0.6

zyme releasewas calculated by comparing enzyme release with zymosan of treated cells (isoproterenol and histamine)

eukocyte

on airways

and

~e~~~c~e

function

studies

Vmaxps (L/se4

3.9 4.2 4.3 4.4 5.4 7.6 5.3 5.1 5.4 5.7 6.0 5.6 4.9 5.4 5.1 5.5 5.1 6.7 10.1 7.2 4.1 4.0 3.8 1.9

of rhinovirus

cytotoxicity

Antibody-dependent cellular cytotoxicity capacity (ADCC) of mononuclear cells and granulocytes was assayed by the method described by Flaherty and co-workers13 using Cr 51-labeled chicken erythrocytes sensitized with

rabbit antichicken erythrocyte antibody as targets. Results are expressed as a corrected cytotoxic index (CCI). In our

laboratory, 8 normal subjects studied 2 months apart had a mean mononuclear CC1 + SD of 47 + 5.13 For granulo-

Vmaxzs He-02 (Llsec)

2.3 2.1 2.2 2.2 2.7 3.8 2.6 2.8 3.2 3.5 4.0 4.0 3.0 3.3 2.2 2.4 2.3 3.9 5.8 4.0 1.4 1.3 1.5 0.6

visa (% vc)*

0 0 22 0 0 0 14 0 0 7 0 0 0 5 19 11 10 7 0 0 19 17 20 23

cytes, the normal mean CC1 ? SD in our laboratory is 85 t 4.13

RESULTS Virus studies Six of seven subjects had RV26 recovered from the nasal washings on at least two occasions in the first week after inoculation (Table I). Four subjects, including Subject 4 who did not shed detectable virus, developed a four-fold or greater rise in serum neutralizing antibody titer to RV16. Signs and symptoms The symptoms produced by this infection were generally mild. All subjects developed upper respiratory symptoms consisting of rhinorrhea, nasal congestion, or pharyngitis. Subjects 1 and 2 experienced a nonproductive cough. Subject 7 corn~la~~ed of moderately severe chest tightness which was accompanied by malaise and fever of 103” F. aximal symptoms occurred on day 4 in Subjects 1, 4, 5, 6, and 7 and in Subjects 2 and 3 on days 1 and 2, respectively.

J. ALLERGY CLIN. MWNOL. FEBRUARY 1978

ush et al.

a

E 111.Percent

inhibition

of lysosomal

enzyme

release

from

granulocytes

lsoproterenol 1O-5

ubject I

2

3

4

5

6

7

0

3 7 21 0 3 7 21 0 3 7 0 3 7 0 3 7 0 3 7 0 3 7 21

ry function

38.5 29.5 30.6 38.7 35.7 24.6 37.5 33.4 31.4 N.D. 37.2 34.7 35.5 32. I 37.1 31.9 35.8 28.0 33. I 29.8 38.4 33.2 24.7 36.8

M

1O-6

Histamine M

34.1 28.0 23.7 24.5 27.6 17.8 31.3 26.7 21.3 N.D. 26.3 27.0 25.9 24.3 28.8 29.3 19.7 21.0 25.3 22.4 21.7 27.6 20.5 28.0

IO-’

M

29.7 22.1 25.0 23.3 18.4 14.5 25.0 16.7 20.3 N.D. 24.4 15.4 19.4 22.4 24.1 18.6 16.1 16.0 18.0 17.6 13.9 24.8 19.1 23.2

1O-5

M

1O-6

39.6 30.2 29.2 40.0 34.8 32.3 38.7 35.0 37.4 N.D. 35.4 30.8 29.3 32.1 35.3 36.3 33.1 37.0 41.8 39.4 37.8 43.3 31.0 38.4

ao-7

39.1 26.5 22.2 31.2 25.3 27.2 32.5 21.7 28.6 N.D. 24.7 25.0 26.7 29.2 29.5 31.9 25.9 24.0 30.1 28.2 27.8 33.8 23.3 28.8

25.3 20.6 16.7 23.4 16.1 17.7 22.5 18.6 22.5 N*D. 17.0 22.2 21.0 22.4 19.7 23.9 15.2 18.0 21.9 21.3 19.5 22.5 16.2 20.8

tests

Except for two subjects (4 and 7), the FEV, varied by less than 10% of predicted throughout the study (Table II). Subject 4 had a 12% fall in predicted FEVl on day 7. The FVC remained within 10% throughout the course of the study. FEF25%-,j% fell by greater than IO% in all but two subjects (5 and 7) on either day 3 or 7. In Subject 7, a 10% decrease in FEFZs~-,,% occurred on day 21. Only Subject 7 de veloped an increased methacholine response ( & FEVr of > 15%). The other showed no response. Vmax,, and VmaxZj while breathing air and the helium-oxygen mixture showed only minimal changes (Table II). Subject 4 had a decrease in flow rates on day 3, while Subject 6 had an increase in flow rates on day 3. The VisoV’s determined at TLC or RV were the same in all cases except for Subject 5 on day 7. His air and He-O, curve vital capacities varied by 4%) resulting in VisoV at TLC of 0% and a VisoV of 11% at

The maximal inhibition of enzyme release w isoproterenol and histamine was achieved at 1QP of each agonist. The three subjects with lower respiratory symptoms (1, 2, and 7) showed a decrease in response to isoproterenol occurring on the study period closest to the day of maximal symptoms (p < 0.05) (Table III). These same subjects also demonstrated a diminished response to histamine at the same time (p < 0.05) (Table I jects with a decreased agonist response, values returned to baseline levels by day 21.

RV.

Six subjects (1, 2,4, 5, 6, and 7) had a decrease in circulating lymphocytes/mm3 during the study (Table IV), although the mean values for days 3 and 7 were not significantly different from the baseline. There was a significant decrease in the number of

The three subjects with lower respiratory symptoms (1, 2, and 7) demonstrated a rise in the VisoV on day 7 (p < 0.01) compared to the 4 subjects who did not have lower respiratory symptoms (Table II).

Leukocyte

and differential

A significant decrease cells (WBCs) was noted (p < 0.02) (Table IV). changes in the differential Lymphocyte

cou

in circulating white blood on day 7 of the infection There were no consistent counts.

subpopulations

Effects of rhinovirus

VOLUivlE 61 NUMBER 2

E-rosetting cells/mm3 (p < 0.05) on days 3 and 7. The percent of EAC-rosetting lymphocytes did not change, but due to a decrease in total circulating lymphocytes, there was a significant decrease in absolute EAC-rosetting cells/mm3 (p < 0.05) on day 7. No consistent changes were noted in Ig-EA cells. ent cellular

cytotoxicity

The mononuclear cell ADCC dropped to subnormal values during the infections (Table IV) (p < 0.02 for the difference between days 1 and 3). There was no significant change in the PMN ADCC. N The illness produced by this experimental infection was extremely mild in Subjects 1 through 5 who received the lower inoculum of virus. Therefore, we administered a higher dose of virus to Subjects 6 and 7. The severity of the symptoms, frequency of recovery of virus from nasopharyngeal washings, and abnormalities in airways or leukocytes did not change appreciably with the higher inoculum. Blair and coworkers4 found no direct relationship between illness and the development of increased frequency dependency of compliance in RV infections, but we found that individuals with lower respiratory symptoms (cough, chest tightness) were the ones who demonstrated airway changes. The absence of significant changes in maximal expiratory flow rates in normal subjects with RV infection is similar to the experience of Fridy and co-workers.6 We found two subjects with elevated VisoV; while VisoV may be quite variable,26 we selected air and helium-oxygen curves with vital capacities within 4% for matching. Only one person developed a positive methacholine test during the course of the infection. This is surprising since viral infections have been reported to result in increased airways irritability.7a * Empey and coworkers* proposed that one mechanism responsible for bronchoconstriction in response to inhaled histamine during upper respiratory infections is the bronchial epithelial damage by the virus itself. They ropose that such damage exposes, and thus “sensitizes,” the rapidly adapting irritant receptors to various stimuli. Although we chose the intranasal route of inoculation to lessen the possibility of lower respiratory tract infection, direct bronchial epithelial damage cannot be entirely excluded as a cause of the exaggerated response. However, other mechanisms may also be operative. Conceivably, the changes in airways function we observed may be related to diminished beta adrenergic response of bronchial smooth muscle. We have no

on airways and ~e~~a~~~e function

direct evidence to support this, but we did observe a significant decrease in the beta-adrenergic response of the granulocytes in the subjects with increases in the volume of isoflow or positive methacboli~e response. These findings are consistent with the previous observations from our laboratory that tbe inhibition of lysosomal enzyme releasell from granulocytes with isoproterenol in asthmatics is decreased during viral respiratory infections provoking an attack of asthma. These studies do not answer the question of whether the decreased beta adrenergic response is the direct consequence of the infection or is secondary to mobilization of endogenous catecholamines that may impair subsequent leukocyte response to isoproterenol. We further noted a decrease in inhibition of lysosomal enzyme release from granulocytes in response to histamine in those subjects who developed peripheral airway changes. Although we did not administer aerosols of histamine to our subjects, asthmatics are known to have an increased sensitivity to inhaled histamine. Busse and Sosmanl” have recently demonstrated a decreased HZ receptor response in granulocytes of asthmatics. It is possible that during a viral infection, a decrease in the feedback inhibitory mechanism modulating release of inflammatory enzymes from PMNs may amplify bronchial inflammation leading to airway obstruction. The implication of decreased mononuclear A capacity during RV infections is not clear. Flaherty and co-workersI found diminished ADCC capacity of mononuclear cells but not PMNs in asthmatics whose attacks were provoked by upper respiratory infection. These studies were performed, however, at a time when the patients were free of infection. The decrease in peripheral blood mononuclear cell A observed in normal persons may be the result distribution, possibly including mobilization of the effector cells to the site of infection. AltemativeIy, the reduction in ADCC could be due to a direct or indirect effect of the virus on mononuclear cell function . It appears that rhinovirus infection decreases the number of circulating T (E rosette forming) lymphocytes and also the mononuclear ADCC. The subjects who developed chest symptoms had evidence of small airway obstruction and decreased beta adrenergic and Hz histamine responses of granulocytes. The difference between the normal and the asthmatic response to respiratory infections appears to be quantitative rather than qualitative. In both normals and asthmatics the airway obstruction is associated with decreased beta adrenergic response. oreover, the bronchial inflammation may be further ampli~ed dur-

.L ALLERGY CLIN. IMMUNOL. FEBRUARY 1978

~~~p~ocyte

subpopulations

ject

1

0 3

21 2

6

0 3 7 21 0 3 I 0 3 7 0 3 7 0

7

0

3

4

5

21 *Nom-al

and ADCC

WBClmm3 (7,250 r l,OOO)*

Lymphslmms (2,500 + 1,500)

7,000 4,200 4,700 7,800 4,600 4,400 3,000 3,700 5,700 6,300 3,700 5,900 8,000 6,900 8,200 7,300 5,900 8,300 7,400 5,600 7,200 7,700 4,500 5,100

2,730 2,688 2,162 2,340 2,070 1,892 1,710 1,443 1,767 1,827 2,035 2,891 2,320 2,829 2,185 1,303 1,168 3,123 2,144 1,637 1,520 1,522 1,386 2,805

% E rosettes (72 r 8)

68 53 54 64 77 72 68 49 67 61 49 69 65 71 65 51 66 71 69 68 64 63 56 62

1,856 1,424 1,167 1,495 L ,594 1,362 1,163 707 1,183 l,i47 997 1,994 1,508 2,008 2,185 1,303 1,168 3,123 2,144 1,637 : ,520 1,552 1,386 1,739

values.

ing virus infections by increased lysosomal enzyme release because of the impairment of the normal feedback inhibitory action of histamine and catecholamines . We wish to acknowledge the technical assistanceof Ann Smith, Jean Surfus, Jean Martin, Marjorie Crandall, William Cooper, and Sue Conklin and to expressappreciation to Nancy Horn and Candace Anderson for their assistance in the preparation

of this manuscript.

FERENCES 1. Minor, T. E., Dick, E. C., Baker, J. W., Ouellette, J. J., Cohen, M., and Reed, C. E.: Rhinovirus and influenza type A infections as precipitants of asthma, Am. Rev. Respir. Dis. 113:149, 1976. 2. Minor, T. E., Dick, E. C., DeMeo, A. N., Ouellette, J. J., Cohen, M., and Reed, C. E.: Viruses as precipitants of asthmatic attacks in children, J.A.M.A. 227~292, 1974. 3. McIntosh, K., Ellis, E. F., Hoffman, L. S., Lybass, T. G., Eller, J. J., and Fulginiti, V. A.: The association of viral and bacterial respiratory infections with excerbations of wheezing in young asthmatic children, J. Pediatr. 82:578, 1973. 4. Blair, H. T., Greenberg, S. B., Stevens, P. M., Bilunos, P. A., and Couch, R. B.: Effects of rhinovirus infection on pulmonary functions of healthy human volunteers, Am. Rev. Respir. Dis. 114~95, 1976.

5. Cate, T. R., Roberts, J. S., Russ, M. A., and Pierce, 3. A.: Effects of common colds on pulmonary functions, Am. Rev. Respir. Dis. 108:858, 1973. 6. Fridy, W. W., Jr., Ingram, R. H., Jr., Hierholzer, J. C., and Coleman, M. T.: Airways function during mild vi& respiratory illness, Ann. Intern. Med. 80:150, 1974. 7. Parker, C. D., Bilbo, R. E., and Reed, C. E.: Methacboiine aerosol as test for bronchial asthma, Arch. Intern. Med. 115~452, 1965. 8. Empey, D. W., Laitinen, L. A., Jacobs, L., Gold, W. W., and Nadel, J. A.: Mechanisms of bronchial byperreactivity in normal subjects after upper respiratory tract infection, Am. Rev. Respir. Dis. 113:131, 1976. 9. Parker, C. W., and Smith, J. W.: Alterations in cyclic adenosine monophosphate metabolism in human bronchial asthma. I. Leukocyte responsiveness to beta-adrenergic agents, J. Clin. Invest. 52:48, 1973. 10. Lee, T. P., Busse, W. W., and Reed, C. E.: Effect of betaadrenergic agonist, prostaglandins, and cortisol on lymphocyte levels of cyclic adenosine monophosphate and glycogen, J.

ALLERGY CLIN. IMMUNOL. 59:408, 1977. 11. Busse, W. W.: Decreased granulocyte response no1 in asthma during upper respiratory infections, Respir. Dis. 115783, 1977. 12. Busse, W. W., and Sosman, J.: Decreased Hz sponse of granulocytes of asthmatic patients, J. 59:1087, 1977. 13. Flaherty, D. K., Martin, J. M., Storms, W. W.,

to isoprotereAm. Rev. histamine reClin. Invest. Kriz,

R. J.,

Effects of rhinovirus

VOLUME 61 NUMBER 2

%

rosettes

k 8)

14. 15.

16.

17.

18.

I9.

EAClmm3 (675 LT 230)

21 19 17 21 20 23 27 25 29 26 25 25 29 18 20 29 18 23

573 511 368

723 673 509 672 740 319 1011

19 20 20 19 16 19

591 481 475 469 369 533

491 414 435 462 361 512 475 509

Surfus, J. E., and Reed, C. E.: Antibody dependent cellular cytotoxicity in asthmatics, J. ALLERGY CLIN. IMMUNOL. 59:48, 1977. Utsinger, P. D.: Lymphocyte changes in viral infections, Ann. Intern. Med. 83:82, 1975. Mangi, R. J., Dwyer, J. M., Neiderman, J. C., andKantor, F. S.: Decreased circulating T cells during viral pharyngitis, Ann. Intern. Med. 81:588, 1974. Wybran, J., and Fudenberg, H. H.: Thymus-derived rosette forming cells in various human disease states; cancer, lymphoma, bacterial and viral infections, and other diseases, J. Clin. Invest. 52: 1026, 1973. Morris, J. F., Koski, A., and Johnson, L. C.: Spirometric standards for healthy, non-smoking adults, Am. Rev. Respir. Dis. 103:57, 1971. D’Alessio, D. J., Peterson, J. A., Dick, C. R., and Dick, E. C.: Transmission of experimental rhinovirus colds in volunteer married couples, J. Infect. Dis. 133:28, 1976. Cooney, M. D., and Kenny, B. E.: Demonstration of dual rbinovirus infection in humans by isolation of different serotypes in human heteroploid (HeLA), and diploid fibroblast cell cultures, J. Clin. Microbial. 52.02, 1977.

% IgE A (14 Ik 4)

on airways and leukocyte function

Mononuclear

88 74 87

0 1 0 6

32 26 34 32

18 4 6 14 17 14 22 1

43 30 23 33 38 32 42

82 80 72 78 74 83 71 74

19 12 6 11 11 18 17

39 32 35 42 39 40 42 43

81 87 81 70 75 74 89 88

22 23 11 7 10

34 51 43 52 25

89 91 89 80 40

20.

Storms, W. W., Dick, E. C., and Busse, W. W.: Intranasal immunization with attenuated live influenza vaccine in asthma, J. ALLERGY CLIN. IMMLJNOL.58~284, 1976. 21. Boyum, A.: Isolation of mononuclear cells and granulocyte from human blood, Stand. J. Clin. Lab. Invest. 97(Supp. 1):77, 1967. 22. Zurier, R. B., Hoffstein, S., and Weismann, 6.: Cytochalasin B: Effect of lysosomal enzyme release from human leukocytes, Proc. Nat’l. Acad. Sci. U.S.A. 70:844, 1973. 23. Busse, W. W.. and Lee, T. P.: Decreased adrenergic responses in lymphocytes and granulocytes in atopic eczema, J.

ALLERGY CLIN. IMMUNOL. 58586, 1976. 24. Fishman, W. H., Kato, K., Anstiss, C. L., and Green, S.: Human serum beta-glucuronidase: Its measurement and some of its properties, Clin. Chim. Acta 15:435, 1967. 25. Caboud, P. G., and Wrobiewski, F.: Calorimetric measurement of lactate dehyrdogenase activity of body fluids, Am. J. Clin. Pathol. 30:234, 1968. 26. Zeck, R. T., Solliday, N. H., and Cugell, D. W.: Variability of the volume of isoflow, Am. Rev. Respir. Dis. 115r392, 1977.