Analysis of nasal secretions during experimental rhinovirus upper respiratory infections

lmmunodeficiency and other clinical immunology

Analysis of nasal secretions during experimental rhinovirus upper respiratory infections

Yasushi Igarashi, MD,’ David P. Skoner, MD,b William J. Doyle, PhD, Martha V. White, MD,” Philip Fireman, MD,b and Michael A. Kaliner, MD” Bethesda, Md., and Pittsburgh Pa.

Background: To determine the underlying mechanisms for rhinovirus-induced nasal secretions, nasal lavage fluids were analyzed during experimental rhinovirus infections. Methods: Twenty patients with allergic rhinitis and 18 nonalletgic control subjects were inoculated with rhinovirus type 39. Nasal lavage was perfomzed before and on days 2 through 7 after viral inoculation, and the lavage fluids were assayed for proteins and mast cell mediators. Results: The secretion of total protein and both plasma proteins (albumin and IgG) and glandular proteins (lactofenin, lysozyme, and secretory IgA) increased after rhinovirus inoculation. Analysis of the specific protein constituents revealed that nasal secretions during the initial response to the rhinovim infection were predominantly due to increased vascular permeability. Allergic subjects tended to have fewer symptoms and more vascular permeability than control subjects, and increased histamine secretion afier rhinovirus inoculation was more frequently seen in the allergy group. Conclusion: Nasal secretions found early in the course of a viral upper respiratory infection are due to increased vascular permeability, whereas glandular secretions predominate later in the infection. (J ALLERGY CLIN IMMUNOL 1993;92:722-31.)

hky words: Alle@ rhinitis, nasal secretion, glandular secretion, rhinovints, vascular

Rhinovirus infections frequently cause the common cold, eliciting local symptoms’ that involve the nasal, pharyngeal, and laryngeal mucous membranes in association with systemic com- plaints. The mechanisms for the mucous membrane responses are not yet clear, however, rhi-

From “the Allergic Diseases Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda; bDepart- ment of Allergy, Immunology and Rheumatology and ‘De- partment of Otolaryngology, Children’s Hospital of Pitts- burgh, University of Pittsburgh School of Medicine.

Supported in part by National Institutes of Health grant AI19262 and MOlRR00084.

Dr. White is a recipient of the Merrell Dow Scholar in Allergy Award.

Received for publication Apr. 30, 1992; revised Feb. 2, 1993; accepted for publication Apr. 16, 1993.

Reprint requests: Michael A. Kaliner, MD, Institute for Asthma and Allergy, 106 Irving St. NW, Suite 125, Wash- ington, DC 20010.

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URI: Upper respiratory infections

novirus infections likely initiate a series of inflammatory reactions involving the release of inflammatory mediators, which in turn cause local responses including increased vascular permeability, glandular secretion, inflammatory cell infiltration, and stimulation of neural pathways.’ Identi- fication of specific rhinovirus-induced mucous membrane responses could lead to innovative approaches for controlling the course and symptoms of the common cold.

Models that incorporate both natural and experimental rhinovirus infections have been used to study the pathophysiology of nasal mucous membrane responses to a rhinovirus infection.‘-g

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No specific morphologic changes can be appreci- ated in the ciliated epithelial cells obtained from nasal scrapings during rhinovirus upper respiratory infection (URI).S The magnitude of lymphocyte infiltration is similar to that seen in normal subjects6 However, the number of polymorphonuclear leukocytes and ciliated epithelial cells shed into nasal isecretions during rhinovirus infections increases concomitantly with a decrease in the shedding of squamous epithelial cells.’ Elevations in certain serum proteins such as albumin, IgG, or IgA have been noted in nasal secretions during viral URI.‘, ’ More recently, the generation of bradykinin in nasal secretions has been demonstrated during both natural and experimental rhinovirus infections.8, 9

Rhinovirus infection shares some symptomatic features with nasal allergic reactions. The proteins found in allergic nasal secretions have been extensively characterized, and analysis has provided insights into their sources. Activation of mast cells after topical allergen provocation results in increased levels of prostaglandin D, (PGD,) and histamine in nasal Iavage fluid.‘” Allergen-induced secretions are also rich in plasma proteins such as albumin and IgG,” as are nasal secretions collected after topical histamine challenge. ‘*, ” It has also been shown that lactoferrin, lysozyme, and secretory IgA (sIgA) are secreted by nasal submucosal glands via a reflex mechanism initiated by antigen challenge,” as well as after cholinergic stimulation or after initi- ation of gustatory reflexes.14-16 The specific cellu- lar source of these glandular products has also been localized by immunohistochemical analysis to the serous ce11.14, Is Thus characterization of the proteins found in nasal secretions can provide information about the source of the increased secretions and some insights into the mediators potentially responsible for triggering the secretions. Secretions rich in plasma proteins reflect increased vascular permeability and implicate ac- tions caused by vasoactive amines (e.g., histamine” or bradykinin’“). Secretions rich in glandular products reflect glandular secretion, commonly induced by cholinergic reflexesI neuropeptides such as gastrin-releasing peptide” and substance P,” or inflammatory mediators such as leukotrienes,” cliymase,22 and eosinophil cationic proteins.2i

Because nasal allergic reactions and rhinovirus infections have similar symptomatic features, it was of interest to know whether the regulatory mechanisms that control nasal secretions in these

two disorders are similar. In the present study, the protein constituents and the inflammatory mediators of rhinovirus-induced nasal secretions were characterized and compared with those in allergic reactions. Moreover, it was also of interest to compare rhinovirus-induced nasal secretory reactions in atopic and nonatopic patients. This population of study subjects has also served as the source for a series of additional parallel studies.24-2h

METHODS Subjects

Twenty subjects with allergic rhinitis (12 men and 8 women, aged 18 to 42 years) and 18 normal control subjects (5 men and 13 women, aged 18 to 44 years) with negligible serum neutralizing antibody titers (~2 dilutions) to rhinovirus trpe 39 were recruited for this study. The diagnosis of allergic rhinitis was based on a history of seasonal rhinitis; concomitant positive skin test results (wheal diameter > 10 mm, intradermal allergen challenge) to ragweed, grass, or tree allergens; and elevated specific serum IgE level ( > 10 IUlml, fluoroallergosorbent test). All but one of the subjects had negative skin test results to house dust, mite, or molds. One subject in the allergy group had a positive skin test result but a negative fluoroallergosorbent test result to house dust and had no history of nasal symptoms in the winter to suggest clinically relevant dust allergy. Subjects in the control group did not have allergy histories and had negative skin test results to the panel of allergen extracts. The study was performed out of the pollen season during March. All subjects re- frained from taking any medications for at least 4 weeks before entry into the study, and no medications were allowed during the trial. No subject was studied within 4 weeks of a URI. The protocol was reviewed and approved by the Institutional Review Board at Chil- dren’s Hospital of Pittsburgh.

Viral challenge

The provocative viral challenges were performed in Pittsburgh as previously described.Z3-Z5 Rhinovirus type 39 (provided by Dr. Jack Gwaltney, Charlottesville, Virginia) was administered as intranasal coarse drops (0.25 ml per nostril) twice during a 30-minute period (total inoculum administered was 100 TCID,, [.50% tissue culture infection dose] per subject). The challenge virus pool was a safety-testedz7 clinical isolate, RV-39, passaged twice in WI-38 human embryonic lung fibroblasts.*’ After the inoculation, subjects were iso- lated in separate hotel rooms for a period of 6 days and 5 nights (days 2 to 7).

Evaluation of infection and illness

Paired viral antibody titers were measured in the serum before and 4 weeks after the challenge to con-

724 lgarashi et al. J ALLERGY CLIN IMMUNOL NOVEMBER 1993

DAYS AFTER INOCULATION

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FIG. 1. Comparison in symptom score and nasal lavage protein secretion during URI between subjects who had the illness and those who did not. A, Total symptom scores (sum of all eight symptom scores) for each day; B, total protein concentration in the nasal lavage. Shown are the scores (mean -t SEM) of 32 subjects in whom the illness developed and 6 subjects in whom it did not. Asterisks indicate significant differences between the two groups (*p c 0.05, **p < 0.01, ***p < 0.001; Mann-Whit- ney U test).

firm the infection” with rhinovirus type 39. Subjects were categorized as infected if they shed virus from nasal washings or had an increase of greater than fourfold in viral antibody titer.

Daily symptom scores were obtained by daily interview. Scores were obtained for sneezing, rhinorrhea, nasal congestion, cough, sore throat, chills, headache, and malaise for 2 days before inoculation and on days 2 through 7 after inoculation. These symptoms were scored on a scale of 0 to 3 (none, mild, moderate, and severe symptoms, respectively). Subjects were defined as ill when the total symptom scores obtained over the 6-day observation period (days 2 to 7) equaled or exceeded 6 points and when the subject had the subjective impression of having a “cold” or had increased rhinorrhea on 3 or more days of 6 (modified criteria of Jackson et a1.30).

Nasal lavage technique

Daily nasal lavages were performed before inoculation and on days 2 through 7 as described previously.’ For each lavage, 5 ml of saline solution was instilled

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FIG. 2. Kinetics of symptom scores and nasal mucus secretion after rhinovirus infection. A, Total symptom scores (sum of all eight symptom scores) for each day; B, rhinorrhea score, C, the weight of expelled nasal mucus secretion (collected in preweighed facial tissues). Mucus was collected for whole days between two lavages. Shown are the scores (mean * SEM) of 17 subjects (allergy group) and 15 subjects (control group). Only sub-

jects in whom the illness developed have been included in the analysis. Asterisks indicate significant differences from the preinoculation level (*p. c 0.05, **p -c 0.01, l **p c 0.001; Wilcoxon signed-rank test). There were no significant differences between the two groups at any single time point (Mann-Whitney U test).

into each naris while subjects held their heads back at a 60-degree angle with their glottides closed, After a period of approximately 30 seconds, lavage fluid was forcibly expelled into a collection vessel. Recovery

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FIG. 3. Kinetics of nasal lavage protein concentrations during URI. The concentration of (A) total protein, (6) albumin, (C) IgG, (D) lactoferrin, (E) lysozyme, and (F) slgA in the nasal lavage. Shown are the concentrations per milliliter of lavage (mean -C SEM) of 17 subjects (allergy group) and 15 subjects (control group). Only subjects in whom the illness developed have been included in the analysis. Asterisks indicate significant differences from the preinoculation level (*p < 0.05, **p < 0.01, ***p < 0.001; Wilcoxon signed-rank test). The differences between the two groups were not significant at any single time point (Mann-Whitney U test).

volume averaged 6 ml in total. Samples were divided into three aliquots: the first was submitted to the virology laboratories for culture, and the remaining two aliquots were stored at - 70” C until they were assayed for proteins or mediators.

Assays Total protein. Total protein concentration in each

sample was measured by the bicinchoninic acid protein assay (Pierce Chemical Co., Rockford, Ill.) with bovine serum albumin as the standard?’ Ten microliters of lavage sample or albumin standard (Pierce Chemical Co.) was placed in polypropylene microtiter plate wells (Dynatech Laboratories, Inc., Chantilly, Va.) in tripli- cate. After 30 minutes of incubation with 200 l~,l of bicinchoninic acid reagent at 37” C, the optical density

at 570 nm was detected with a microplate reader (Dynatech Laboratories, Inc.). The protein concentration in samples was determined from the regression analysis of the standard curve. The assay is accurate over a range of 15 to 2000 &ml. Preliminary experi- ments showed that similar results were obtained by using either the bicinchoninic acid protein assay or the Lowry method3’ (data not presented).

Albumin. Albumin was measured by a competitive ELISA, modified from a previously described method.14 The concentration of albumin was determined from standard curves, The assay is accurate between 1 and 100 &#nl.

Lactofetin, IgG, and s&A. Lactoferrin, IgG, and sIgA were measured by modified noncompetitive ELI- SAs as previously described.13, 14, 33 The assays for lactoferrin, IgG, and sIgA are accurate between 1 and 100


0 I PRE- 2 3 4 5 6 7

DAYS A,FTER INOCULATION


FIG. 4. The relative concentration of plasma proteins (albumin plus IgG) and glandular proteins (lactoferrin plus lysozyme plus slgA) to total protein in the nasal lavage during URI. A, The relative concentration of plasma proteins (aggregate of percents of albumin and IgG; 6, the relative concentration of glandular proteins (aggregate of percents of lactoferrin, lysozyme, and slgA). Shown are the relative concentrations (mean f SEM) of 17 allergic subjects and 15 control subjects. Only subjects in whom the illness developed have been included in the analysis. Asterisks indicate significant differences from the preinoculation level (*p c 0.05, **p < 0.01, ***p < 0.001; Wil- coxon signed-rank test). The differences between the two groups were not significant at any single time point (Mann-Whitney U test).

@ml, 1 and 100 @ml, and 3 and 400 @ml, respectively.

Lysozyme. Lysozyme activity was measured by a turbidimetric assay based on the enzymatic hydrolysis of bacterial cell walls as previously described.“~ 34 The range of the assay was between 1 and 100 &ml.

Percents of albumin, IgG, lactofenin, lysozyme, and s&4. Percents of individual proteins to total protein were calculated by dividing the concentration of respective proteins by that of total protein and multi- plying by l&I%.

Histamine and PGD, Histamine and PGD, were measured by competitive radioimmunoassays as described.35, 36 The assay range was between 0.02 and 16 @ml for histamine and between 31 and 2000 pg/ml for PGD,.

Statistics

Because most of the data were not normally distrib- uted, statistical differences between different time points were examined by the Wilcoxon signed-rank test. Differences between the two groups (allergy and control) were analyzed by the Mann-Whitney U test. The correlations between protein and inflammatory mediators were analyzed by Spearman’s rank correlation test.

RESULTS Effectiveness of viral inoculation

All 38 subjects (20 allergic and 18 control) inoculated with rhinovirus type 39 were infected as determined by viral specific antibody titers and viral sheddings into the nasal washings. Thirty- two (17 allergic and 15 control) subjects were determined to have become ill by modified Jack- son’s criteria. In the group in which illness developed, both total symptom scores and total protein concentration in the nasal lavage dramatically increased after rhinovirus inoculation, whereas the levels were significantly lower in the group in which the illness did not develop (Fig. 1). On the preinoculation day, the “ill” group had slightly higher symptom scores and total protein concentration values in the nasal lavages than the “non- ill” group, although these differences were not significant. Although the kinetics of nasal protein secretion were similar, including the data from the “non-ill” subjects’ in the analysis might reduce its sensitivity; therefore, the results obtained from the “non-ill” group were excluded from the analysis.

Changes in symptom scores

In both the allergy and the control groups, total symptom scores were significantly higher than preinoculation scores on days 2 through 7 (Wil- coxon signed-rank test). The allergy group and the control group had the highest scores on days 2 and 3, respectively (Fig. 2, A). Symptom scores were not obtained by interview on day 1; however, patients recorded self-scored symptom diaries on days 0 through 13. The peak day of symptoms in the allergy group was actually day 2, since self- recorded scores on day 2 were higher than on day 1 (data not presented). Symptoms of rhinorrhea paralleled total symptom scores (Fig. 2, B). The control group complained of slightly more syrnp- toms than did the allergy group, although the difference was not significant at any individual time point (Mann-Whitney U test). To provide a measure of secretion production, subjects expelled all nasal secretions into preweighed tissues and sealed expended tissues in plastic baggies from days 2 through 7. Weights of mucus secre-

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0 0 PRE- 2-4.DAYS PRE- 2-4 DAYS

INOCULATION AFTER INOCULATION INOCULATION AFTER INOCULATION

FIG. 5. Changes in histamine concentrations in the nasal lavage after rhinovirus inoculation. Shown are the preinoculation levels and the maximum levels in 2 to 4 days after rhinovirus inoculation in individual subjects; A, allergy group, B, control group. Only subjects in whom the illness developed have been included in the analysis. Cross bars represent the mean histamine concentration of 17 subjects (allergy group) and 14 subjects (control group). One subject (control group) who showed a disproportionally high histamine level was excluded from the calculation of the mean. Asterisks indicate significant differences from the preinoculation level (***p < 0.001; Wilcoxon signed-rank test). The change in the control group was not significant regardless of whether the outlier individual was included. The differences between the two groups were significant on the preinoculation day (p < 0.05) but not after the viral inoculation (Mann-Whitney U test).

tions were obtained by subtracting the weight of the bag itself and unused tissues from the weight of bags that contained used tissues. In both groups th.e weight of blown secretions peaked between days 3 and 4 (Fig. 2, C). The allergy group secreted equal (or slightly more) blown secretions (differences not significant) than the control group.

Changes in nasal protein secretions

Analysis of nasal lavage fluid revealed that the allergy group had higher baseline protein levels than the control group, although the differences were not significant (Fig. 3). In both groups the concentra.tion of total protein increased after rhinovirus inoculation (Fig. 3, A). The changes were very rapid in the allergy group, peaking on day 3 and returning to the preinoculation level on day 7. In contrast, the changes in the control group were milder than in the allergy group, peaking on day 4 and remaining elevated for a longer period. The total protein concentration on the peak day was 5 to 7 times higher than that on the preinoculation day. When the magnitude of total protein concentration in the nasal lavages between subjects with allergic rhinitis and control subjects was com-

pared, allergic subjects tended to secrete more protein on their peak day and less protein on days 6 to 7 than control subjects. However, there were no significant differences between the two groups at any time point (Mann-Whitney U test).

The concentrations of both plasma proteins (albumin and IgG) and glandular proteins (lactoferrin, lysozyme, and sIgA) increased during the rhinovirus cold (Fig. 3, B-F). The increase in the earlier stage (days 2 to 3) was more dramatic in plasma proteins than in glandular proteins: an increase of lo- to 15fold versus an increase of 1.5 to 5-fold, respectively. The decrease in the later stage was more noticeable in plasma proteins than in glandular proteins; the concentrations of lactoferrin and sIgA were rather higher on days 5 to 6 than on days 3 to 4. The relative concentration of all measured plasma proteins to the total protein (the aggregate of percents of albumin and IgG) increased from 15% (before inoculation) to 45% (day 3), and decreased to 20% on day 7 (Fig. 4). On the other hand, the relative concentration of all measured glandular proteins to the total protein (the aggregate of percents of lactoferrin, lysozyme, and s&A) decreased from 40% (before inoculation) to 20%


(day 3) and increased to 45% on day 7. As with total protein, the allergy group tended to secrete these proteins more in the early stage and less in the later stage than the control group. However, most of the differences between the two groups were not significant (Mann-Whitney U test).

Inflammatory mediators

There was a substantial amount of histamine in the preinoculation nasal lavages (Fig. 5). Interest- ingly, the control group showed a significantly. higher level of histamine than the allergy group on the preinoculation day (p < 0.05; Mann-Whit- ney U test). In the allergy group the histamine level in the nasal lavage was increased after the rhinovirus inoculation in 13 of 17 subjects, whereas in the control group the histamine level was increased in only 4 of 15 subjects (Fig. 5). There was a single subject in the control group who secreted an extremely high amount of histamine in the nasal lavage throughout the observation period (Fig. 5). This subject did not demon- strate a commensurate disproportionally high secretion of proteins. The concentration of PGD, in the nasal lavage increased in only three subjects in each group and mostly became undetectable during the illness (data not presented). Those who demonstrated increased PGDp secretion did not necessarily show increased histamine secretion. To examine the relationship between histamine and nasal secretion, the correlation between histamine and total protein or between histamine and either of the plasma proteins was analyzed. Because each individual’s sensitivity to histamine for inducing vascular leakage may vary, Spear- man’s analysis was performed individually; then the individual correlation coefficients were averaged for all subjects. The correlation between histamine and total protein secretion was weak (r = 0.18 + 0.11 in the allergy group, r = -0.05 + 0.11 in the control group; not significant). The correlations between histamine and the plasma protein secretions were also weak (similar r values as in the case of total protein; not significant; data not presented).

DISCUSSION

Experimental rhinovirus inoculation has several advantages over the study of natural infections. The exact time of infection can be defined, uni- formity of infection can be maximized, and other environmental effects can be minimized by isolat- ing subjects. In this study all subjects who received rhinovirus inoculation were infected, and

more than 80% exhibited the criteria for clinical illness. The kinetics of total protein secretion were similar in the “ill” group and the “non-ill” group, suggesting that the “non-ill” group repre- sents very mild infection.

Rhinorrhea, as well as sneezing and nasal congestion, is prominent in rhinovirus infections. The kinetics of the rhinorrhea score were similar to those of the total symptom score. Curiously, un- like other studies,37 the kinetics of nasal mucus secretion were slightly different from those of symptom scores. The mean rhinorrhea symptom score reached its peak level on day 2; however, the quantity of protein secreted rose more slowly, peaking on day 3. It is conceivable that sometimes the subjects’ subjective evaluation of rhinorrhea may be biased by their feelings of general illness or well-being. Interestingly, the allergy group, which actually demonstrated equivalent or greater nasal secretion than the control group, tended to have fewer subjective symptoms. One explanation for this finding could be that because allergic patients frequently experienced nasal symptoms, they might become more tolerant of those symptoms and thus report lower symptom scores than the control subjects.

To characterize the source of nasal secretions stimulated by rhinovirus infection, plasma (albumin and IgG) and glandular (lactoferrin, lysozyme, and s&A) proteins were measured in the nasal lavage fluid. The plasma proteins showed marked increases in the early stages, peaking on day 3, along with the peak in total protein secretion. The increase in glandular proteins was of lower magnitude than that of the plasma proteins, and the kinetics of glandular protein secretions varied among three different proteins; secretion of lactoferrin and lysozyme seem to have peaked slightly earlier than that of s&A. Butler et al.’ reported that IgA, which consists predominantly of secretory IgA,38 rose after the illness had subsided. They presumed that the rise in IgA was due to increased local synthesis of virus-specific antibodies. The difference in kinetics of secretion between sIgA and other glandular proteins may be explained by the increased local sIgA production. Another possibility is that because polymorphonuclear cells contain lactoferrin3’ and lysozyme40 and are found in the nasal secretions of patients with rhinovirus infection,’ a small portion of the lactoferrin and lysozymes secreted into the nasal lavage may be derived from polymorphonuclear cells. However, the lactoferrin content of a neutrophil (6 to 8 Fg

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lactoferrin406 neutrophils4*) and the neutrophil count in nasal lavages during a URI ( < 10” neutrophils/ml lavage fluidg) do not account for the levels of lactoferrin seen in this study.

Because in this way of collecting nasal lavage, an increas.ed nasal fluid turnover (in situations such as that of the present study) would lead to underestimations for the magnitude of actual nasal secretion, we used the relative concentration of individual proteins to total protein as an index of the source of the secreted protein. It is affected neither by the turnover rate nor by the recovery ratio. After methacholine nasal challenge, although the concentrations of both plasma and glandular proteins in the nasal lavage increase, the relative concentration of albumin (plasma protein) to total protein remains unchanged from the baseline value, whereas the relative concentration of :sIgA (glandular protein) to total protein increases.‘* On the other hand, after histamine nasal challenge, the relative concentration of albumin increases, whereas the relative concentration of sIgA decreases.“’ If patients with allergic rhinitis are challenged with allergen, again the concentrations of both plasma and glandular proteins in the nasal lavage increase, but in this case the relative concentration of albumin increases and the relative concentration of sIgA remains unchanged from the baseline value.” In the present study, the relative concentration of plasma proteins significantly increased during the rhinovirus. cold, whereas the relative concentration of glandular proteins decreased significantly. This result suggests that protein secretion during a rhinovirus cold is predominantly due to increased vascular permeability. Later in the resolution of the URI, the pattern of ,protein secretion changed; glandular secretions rather predomi- nated.

Because nasal secretions during the initial phase of rhinovirus infections are predominantly derived from vascular leakage, the possibility that inflammatory mediators released from mast cells might in part be responsible for the increased vascular permeability was considered. In an at- tempt to determine whether mast cell or basophil activation might contribute to rhinovirus-induced secretions, histamine and PGD, levels were measured. Histamine was present in nasal lavages obtained before rhinovirus inoculation. The control group had higher baseline histamine levels than the allergy group, but this difference was not reflected for enhanced baseline protein secretions. Contrary to previous observations,g, ” the

secretion of histamine increased during the rhinovirus infection in some individuals (mainly in the allergy group). Histamine in nasal secretions during a URI has previously been measured by the fluorometric assay,43 and the lavage interval used was shorter than that used in the present study. Therefore at least in some patients in this study, histamine may be released during rhinovirus infection. On the other hand, the secretion of PGD, failed to increase after rhinovirus inoculation in either group. These data suggest two possibilities. (1) If mast cells are stimulated during rhinovirus infection, non-IgE-mediated secretagogues, such as substance P,” bradykinin,45 or inflammatory cell-derived histamine-releasing fac- tors,* may be involved. These secretagogues se- lectively induce histamine release but not eicosanoid generation from mast cells.“-46 (2) The other possibility is that the source of histamine is basophils. Basophils release histamine but not PGD,.47 However, in either case, histamine is unlikely to play a major role in inducing increased vascular permeability during rhinovirus infections for several reasons. First, increased secretion of histamine occurred only in a subgroup of viral- inoculated subjects, whereas all of the subjects secreted increased proteins. Second, extraordi- narily high histamine levels in a single control subject were not associated with increased secretion of plasma proteins in that individual. Third, the amount of histamine in nasal secretions did not correlate with the level of protein secretion. Proud et al.’ reported a marked increase in kinins but no increase in histamine during rhinovirus infections, implicating kinins as a possible cause of the increased vascular permeability seen during a rhinovirus URI. Although the predominant response to kinins involves activation of kinin p2- receptors in the vascular bed with resultant vascular permeability,4x* 4g kinins also have been demonstrated to induce reflex glandular secretion.50% 5’ Although we did not measure kinins, our data are consistent with kinin generation.

From the data reported herein, it is clear that nasal secretions during the initial stages of a URI are predominantly of vascular origin, since the percentage of albumin and IgG to total protein in the nasal lavages increased after virus inoculation and approached that of plasma. It is likely that vascular leakage contributes to the resolution of a URI by washing out viruses or by facilitating the transport of inflammatory cells and immunoglob- ulins to the site of infection. Persson5’ has recently presented data suggesting that the initial


vascular permeability phase of inflammation is an important stage in host defense, and these data confirm that the initial inflammatory response is indeed one of plasma protein exudation in a rhinovirus URI. Glandular protein secretion also increased during the rhinovirus infection. Of the glandular proteins measured, sIgA was secreted later than others. This finding may reflect increased synthesis of specific antiviral antibodies, as suggested by Butler et al.’ It is also possible that antimicrobial proteins such as lactoferrin and lysozyme secreted by the submucosal glands may be beneficial for recovery.

In a cold, as in other nasal inflammatory responses, the secretion of plasma and glandular proteins likely help to resolve the infection and are orchestrated to restrict the progression of the infection.

REFERENCES

1. Gwaltney JM Jr, Hendley LO, Simon G, Jordan WS Jr. Rhinovirus infections in an industrial population. I. The occurrence of illness. N Engl J Med 1966;275:1261-8.

2. Turner RB, Hendley JO, Gwaltney JM Jr. Shedding of infected ciliated epithelial cells in rhinovirus colds. J Infect Dis 1982;145:849-53.

3. Remington JS, Vosti KL, Lietze A, Zimmerman AL. Serum proteins and antibody activity in human nasal secretions. J Clin Invest 1964;43:1613-24.

4. Hamory BH, Hendly JO, Simon G, Jordan WS Jr. Rhi- novirus growth in nasal polyp organ culture. Proc Sot Exp Biol Med 1977;155:577-82.

5. Douglas RG Jr, Alfred BR, Couch RB. Atraumatic nasal biopsy for studies of respiratory virus infection in volunteers. In: Hobby GL, ed. Antimicrobial agents and che- motherapy-1968. Bethesda: American Society for Micro- biology, 1968:340-3.

6. Winther B, Innes DJ, Brastsch J, Hayden FG. Lymphocyte subsets in the nasal mucosa and peripheral blood during experimental rhinovirus infection. Am J Rhino1 1992;6: 149-56.

7. Butler WT, Waldmann TA, Rossen RD, Douglas RG Jr, Couch RB. Changes in IgA and IgG concentrations in nasal secretions prior to the appearance of antibody during viral respiratory infection in man. J Immunol 197O;lOS: 584-91.

8. Proud D, Naclerio RM, Gwaltney JM, Hendley JO. Kinins are generated in nasal secretions during natural rhinovirus colds. J Infect Dis 1990;161:120-3.

9. Naclerio RM, Proud D, Lichtenstein LM, et al. Kinins are generated during experimental rhinovirus colds. J Infect Dis 1988;157:133-42.

10. Naclerio RM, Meier HL, Kagey-Sobotka A, et al, Medi- ator release after nasal airway challenge with allergen. Am Rev Respir Dis 1983;128:597-602.

11. Raphael GD, Igarashi Y, White MV, Kaliner MA. The pathophysiology of rhinitis. V. Sources of protein in allergen-induced nasal secretions. J ALLERGY CLIN IMMIJNOL 1991;88:33-42.

12. Raphael GD, Meredith SD, Baraniuk JN, Druce HM,

Banks SM, Kaliner MA. The pathophysiology of rhinitis. II. Assessment of the sources of protein in histamine- induced nasal secretions. Am Rev Respir Dis 1989;139: 791-800.

13. Meredith SD, Raphael GD, Baraniuk JN, Banks SM, Kaliner MA. The pathophysiology of rhinitis. III. The control of IgG secretion. J ALLERGY CLIN IMMUNOL 1989; 84:920-30.

14. Raphael GD, Druce HM, Baraniuk JN, Kaliner MA. Pathophysiology of rhinitis. 1. Assessment of the sources of protein in methacholine-induced nasal secretions. Am Rev Respir Dis 1988;138:413-20.

15. Raphael GD, Jeney EV, Baraniuk JN, Kim I, Meredith SD, Kaliner MA. Pathophysiology of rhinitis. Lactoferrin and lysozyme in nasal secretions. J Clin Invest 1989;84:1528-35.

16. Raphael GD, Hauptschein-Raphael M, Kaliner MA. Gus- tatory rhinitis: a syndrome of food induced rhinorrhea. J ALLERGY &IN IMMUNOL 1989;83:110-4.

17. Okuda M, Ohtsuka H, Sakaguchi K, Watanabe T. Nasal histamine sensitivity in allergic rhinitis. Ann Allergy 1983; 51:51-s.

18. Bisset GW, Lewis GP. A spectrum of pharmacological activity in some biologically active peptides. Br J Pharma- co1 Chemother 1962;19:168-82.

19. Baraniuk JN, Lundgren JD, Goff J, et al. Gastrin releasing peptide (GRP) in human nasal mucosa. J Clin Invest 1990;85:998-1005.

20. Baraniuk JN, Lundgren JD, Okayama M, et al. Substance P and neurokinin A in human nasal mucosa. Am J Respir Cell Mol Biol 1991;4:228-36.

21. Marom Z, Shelhamer JH, Bach MK, Morton DR, Kaliner MA. Slow-reacting substances, leukotrienes C, and D,, increase the release of mucus from human airways in vitro. Am Rev Respir Dis 1982;126:449-51.

22. Sommerhoff CP, Caughey GH, Finkbeiner WE, Lazarus SC, Basbaum CB, Nadel JA. Mast cell chymase: a potent secretagogue for airway gland serous cells. J Immunol 1989;142:2450-6.

23. Lundgren JD, Davey RT Jr, Lundgren B, et al. Eosinophil cationic protein stimulates and major basic protein inhibits airway mucus secretion. J ALLERGY CLIN IMMLJNOL 1991;87: 689-98.

24. Skoner D, Whiteside T, Herberman R, Kiss J, Doyle W, Fireman P. Effect of rhinovirus 39 (RV) infection on immune/inflammatory cell functions [Abstract]. J ALLERGY

CLIN IMMUNOL 1991;87:306. 25. Doyle WJ, Skoner DP, Fireman P, et al. Rhinovirus 39

infection in allergic and nonallergic subjects. J ALLERGY CLIN hMUNOL 1992;89:968-78.

26. Fireman P, Skoner DP, Doyle WJ. Effect of rhinovirus (RV) 39 infection on responses of the lower airway and the leukocyte [Abstract]. Am Rev Respir Dis 1991;143: A420.

27. Knight V. The use of volunteers in medical virology. In: Melnick JL, ed. Progress in medical virology. Basel, Switzerland: Karger, 1964;6:1-26.

28. Gwaltney JM, Jordan W. Rhinovirus and respiratory illness in university students. Am Rev Respir Dis 1966;93: 362-71.

29. Humparian W. Rhinovirus. In: Lennette EH, Schmidt NJ, eds. Diagnostic procedures for viral, rickettsial and chlamydial infections. 5th ed. Washington DC: American Public Health Association, 1979:535-75.

30. Jackson GG, Dowling HF, Spiesman IG, Boand AV. Transmission of the common cold to volunteers under

J ALLERGY CLIN IMMUNOL VOLUME 92, NLJMBER 5

lgarashi et al. 731

controlled conditions. I. The common cold as a clinical entity. Arch Intern Med 1958;101:267-78.

31. Smith PK, Krohn RI, Hermanson GT, et al. Measurement of protein using bicinchoninic acid. Anal Biochem 1985; 150:76-85.

32. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75.

33. White MV, Kaliner MA. Neutrophils and mast cells. I. Human neutrophil-derived histamine-releasing activity. J Immunol 1987;139:1624-30.

34. Metcalf JA, Gallin JI, Nauseef WM, Root RK. Lysozyme. In: Laboratory manual of neutrophil function. New York: Raven Press, 1986:149-50.

35. McBride P, Bradley D, Kaliner M. Evaluation of a radioimmunoassay for histamine measurement in biologic fluids. J AL.LERGY CLIN IMMUNOL 1988;82:638-45.

36. Granstrom E, Kindahl H. Radioimmunoassay of prostaglandin and thromboxanes. In: Frolich JC, ed. Advances in prostaglandin and thromboxane research. New York: Raven Press, 1978;5:119-210.

37. Parekh HH, Cragun KT, Hayden FG, Hendley JO, Gwalt- ney JM .Jr. Nasal mucus weights in experimental rhinovirus infection. Am J Rhino1 1992;6:107-10.

38. Rossen RD, Kasel JA, Cough RB. The secretory immune system: its relation to respiratory viral infection. In: Melnick JL, ed. Progress in medical virology. Basel, Switzerland: Karger, 1971:194-238.

39. Hegnhoj J, Schaffalitzky de Muckadell OB. An enzyme- linked immunosorbent assay for measurements of lactoferrin in duodenal aspirates and other biological fluids. Stand J Clin Invest 1985;45:489-95.

40. Klockars M, Reitamo S. Tissue distribution of lysozyme in man. J Histochem Cytochem 1975;23:932-40.

41. Metcalf JA, Gallin JI, Nauseef WM, Root RK. Quantita- tive assay of lactoferrin (ELISA assay). In: Metcalf JA, Gallin Jl:, Nauseef WM, Root RK, eds. Laboratory manual of neutrophil function. New York: Raven Press, 1986: 153-5.

42. Egglestcsn PA, Hendley JO, Gwaltney JM Jr. Mediators of immediate hypersensitivity in nasal secretions during nat-

ural colds and rhinovirus infection. Acta Otolaryngol (Stockh) 1984;(suppl 413):25-35.

43. Siraganian RP. An automated continuous-flow system for the extraction and fluorometric analysis of histamine. Anal Biochem 1975;57:383-94.

44. Levi-Schaffer F, Schalit M. Differential release of histamine and prostaglandin D, in rat peritoneal mast cells activated with peptides. Int Arch Allergy Appl Immunol 1989;90:352-7.

45. Cohan VL, MacGlashan DW Jr, Warner JA, Lichtenstein LM, Proud D. Mechanisms of release from human skin mast cells upon stimulation by the bradykinin analog, [&go-Hyp3-,Phe’]bradykinin. Biochem Pharmacol 1991; 41:293-300.

46. Igarashi Y, Lundgren JD, Doerfler ME, Shelhamer JH, Kaliner MA, White MV. Human neutrophil-derived histamine-releasing activity (HRA-N) causes the release of serotonin but not arachidonic acid metabolites from rat basophilic leukemia cells. J ALLERGY CLIN IMMUNOL 1992; 89:1085-97.

47. MacGlashan DW Jr, Schleimer RP, Peters SP, et al. Comparative studies of human basophils and mast cells. Fed Proc 1983;42:2504-9.

48. Baraniuk JN, Lundgren JD, Mizoguchi H, et al. Bradyki- nin and respiratory mucous membranes: analysis of bradykinin binding site distribution and secretory responses in vitro and in vivo. Am Rev Respir Dis 1990;141:706-14.

49. Holmberg K, Bake B, Pipkorn U. Vascular effects of topically applied bradykinin on the human nasal mucosa. Eur J Pharmacol 1990;175:35-41.

50. Lewis GP. Kinins in inflammation and tissue injury. In: ErdBs EG, ed. Handbook of experimental pharmacology. Bradykinin, kallidin, and kallikrein. Berlin: Springer-Ver- lag, 1970;25:516-30.

51. Garcia Leme J. Bradykinin-system. In: Vane JR, Ferreira SH, eds. Handbook of experimental pharmacology. In- flammation. Berlin: Springer-Verlag, 1978;50:464-522.

52. Persson CGA. Plasma exudation in tracheobronchial and nasal airways: a mucosal defense mechanism becomes pathogenic in asthma and rhinitis. Eur Respir J 1990;3: 652-1s.

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Analysis of nasal secretions during experimental rhinovirus upper respiratory infections

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