Abstract
Knowledge of the virus-induced immune response is important in understanding the pathophysiology of respiratory virus infections. Data on the cellular immune response is still limited and based mainly on experimental studies. Natural colds may differ in their pathophysiology from experimentally induced ones. To evaluate the inflammatory cell responses in the upper respiratory tract during natural colds we counted the number of lymphocytes, mast cells and macrophages in the nasal mucosa. Nasal biopsies were taken from 22 adult volunteers during the acute (2–4 days of symptoms) and convalescent phases (day 21) of the cold, and the numbers of cells were counted with immunohistochemical methods. Viral aetiology was identified in 14 (64%) subjects by using viral isolation, antigen detection and rhino-polymerase chain reaction assays. The number of T lymphocytes was increased in the nasal epithelium and that of T and B lymphocytes and mast cells in the subepithelial layer in the acute phase compared to the convalescent phase. Intraepithelial T lymphocyte counts were significantly higher in the subjects who had a proven viral infection or a finding of pathogenic bacteria in the nasopharynx compared to the subjects without such findings (P = 0·005 and P = 0·04, respectively). Contrary to the earlier experimental studies, we found that viruses cause accumulation of T and B lymphocytes and mast cells during the first days of a symptomatic naturally acquired respiratory infection.
Keywords: cellular immunity, common cold, rhinitis, virus
INTRODUCTION
Viral respiratory tract infections are usually mild and self-limiting, but may lead to bacterial complications, such as sinusitis and pneumonia, and may cause airway obstruction in asthmatic children and adults [1,2]. Knowledge of the virus-induced immune response is important in understanding the pathophysiology of respiratory virus infections. Data on the cellular immune response, which is an important part of the host response in the upper airways during respiratory virus infections, is still limited and based mainly on experimental studies of infections induced in volunteers using one specific virus.
The inflammatory cellular changes in the airway during a viral infection have most often been studied in the nasal mucosa and in nasal secretions due to their good accessibility. An increase in neutrophil counts in the nasal mucosa and nasal secretions has been found in experimental rhinovirus infections during the first days of infection [3–5]. The numbers of lymphocytes, macrophages and mast cells have not increased during experimental rhinovirus colds [3,6]. Yet, the concentrations of several proinflammatory cytokines, IL-1β, IL-6 and RANTES, which are T and B lymphocyte activators, have been shown to be elevated in nasal lavage during natural colds [7]. Increased numbers of T lymphocytes have been seen in nasal secretions [5] and the lower airway epithelium [8] coincident with increased airway responsiveness.
We examined the lymphocyte, macrophage and mast cell counts in the nasal mucosa using immunohistochemical methods during natural colds of 2–4 days' duration. The viral aetiology was studied by means of virus isolation, antigen detection and rhino-polymerase chain reaction (PCR) methods. In addition, we explored whether the finding of pathogenic bacteria in the nasopharynx had any effect on the cellular responses.
MATERIALS AND METHODS
Subjects
Twenty-five subjects were recruited during two periods between 1 February and 15 May 1996 and 15 August and 31 December 1996 by requests for adult volunteers with an acute community-acquired cold by advertising in a newspaper distributed in Oulu, a city in Finland with about 120 000 inhabitants. The inclusion criteria were: duration of symptoms of acute common cold for 2–4 days, presence of nasal symptoms, no prior sinusitis episodes or nasal polyps and no ongoing antibiotic treatment. The three subjects who were classified as atopic, because they had at least one positive (≥3 mm) skin prick test among the 18 common inhalants tested (Prick-Lancett, Ewo Care AB, Gislaved, Sweden) [9], were excluded, leaving 22 subjects in the series. The Ethical Committee of the University of Oulu found the study protocol ethically acceptable, and written informed consent was obtained from all subjects.
The subjects were examined at enrolment (acute phase) and again after 21 days (convalescent phase). Background variables were recorded on a questionnaire. The subjects rated the following 10 symptoms for 3 days using a scale from 0 (not present) to 10 (very severe): runny nose, nasal stuffiness, sneezing, sore throat, facial pain, cough, fatigue or lethargy, muscle aches, chills and headache. The sum of the individual scores was used as the total symptom score.
The viral aetiology of the cold was evaluated by antigen detection (adeno, respiratory syncytial (RS), influenza A and B, parainfluenza 1–3 viruses) [10] from nasal mucus and virus isolations (adeno, RS, influenza A and B, parainfluenza 1–3 and enteroviruses) [10] and rhino-PCR from nasopharyngeal swabs [11–13]. Several methods were used to maximize virus detection. The samples of nasopharyngeal swaps were treated with a RT-PCR test for rhino- and enteroviruses, and the product was hybridzed with a rhinovirus-specific probe. Specimens for aerobic bacterial cultures were taken from the nasopharynx. The number of lymphocytes in the peripheral blood was counted.
Nasal biopsies
Nasal mucosal biopsies were taken in the acute and convalescent phases from the inferior turbinate following local application of 4% lidocain [14]. The specimens were fixed in formalin and embedded in paraffin, and 4 µm thick sections were stained with immunohistochemical methods as described previously [15]. In brief, for mast cell staining, the sections were treated with trypsin (1 g/l; 10 min at 37°C) and incubated with the mouse monoclonal antibody against mast cell tryptase (Clone AA1; Dako, Glostrup, Denmark). For CD3, rabbit polyclonal antiserum (Code A 0452; Dako) was used after boiling the sections in 10 ml/l citrate buffer for 20 min. For CD20 detection, the sections were incubated with monoclonal antibodies (Clone L26; Dako). For CD68 staining, monoclonal antibody (Clone PG-M1; Dako) was used after treatment with pepsin (0·4% Merck Darmstad, Germany) at 37°C for 2 h. Bound antibodies were detected by the avidin–biotin method (Dako). For negative control stainings, the primary antibody was replaced with phosphate-buffered saline.
All histological analyses were performed by an investigator who was blinded to all the other information. Cells in the epithelium and in the mucosa beneath the basement membrane of the epithelium were counted in a minimum of three consecutive fields and expressed as cells/mm2. To assess the repeatability of the cell count, 10 randomly selected classes were counted twice blindly, and a good correlation between assays was obtained (correlation coefficient 0·8).
Of the 22 subjects, 20 had biopsies evaluable on microscopy in the acute phase and 22 in the convalescent phase. Epithelium was missing in four (20%) of the subjects during the infection and in four (18%) during the convalescent phase. The absence of the epithelium was not associated with any of the clinical variables or subepithelial cell counts (data not shown).
Statistical analysis
Summary statistics are expressed as medians and ranges. Data were analysed using Wilcoxon ranked-pairs analysis for paired data and the Mann–Whitney U-test for unpaired data with two groups and the Kruskal–Wallis analysis of variance for unpaired data with more groups. Correlation coefficients were calculated using Spearman's rank correlation.
RESULTS
Viral aetiology was verified in 14 (64%) of the subjects (Table 1). The subjects with proven viral infection were significantly younger than those without (P = 0·03), but otherwise there were no differences in the subject characteristics.
Table 1.
Subject characteristics of 22 adults having natural colds*
| Proven viral infection (n = 14) | Non-proven viral infection infection (n = 8) | |
|---|---|---|
| Mean (s.d.) age in years | 34 (11) | 46 (13) |
| Women | 9 (64) | 7 (87) |
| Current smokers | 8 (57) | 2 (25) |
| Median (range) symptom scores | 122(61–164) | 167(76–319) |
| Viral aetiology† | ||
| ″No virus detected | 8 (100) | |
| ″Rhinovirus | 6 (43) | |
| ″Adenovirus | 4 (29) | |
| ″Parainfluenza type 1, 3 | 2 (14) | |
| ″Enterovirus | 1 (7) | |
| ″Rhino and respiratory syncytial virus | 1 (7) | |
| Pathogenic bacteria in nasopharynx | ||
| ″S. pneumoniae | 2 (12) | |
| ″H. influenzae | 3 (21) | |
| ″M. catarrhalis | 1 (7) | 4 (50) |
| ″Overall | 6 (43) | 4 (50) |
Numbers of subjects and percentages unless otherwise stated.
Detected by means of viral culture, antigen detection and rhino-polymerase chain reaction.
T lymphocytes were the most frequent mononuclear cells in the epithelium, and their amount was significantly higher in the acute phase than in the convalescent phase (Figs 1 and 2, Table 2). The numbers of mast cells, B lymphocytes and macrophages in the epithelium were similar in the acute and convalescent phases. The epithelial T lymphocyte counts in the acute phase did not correlate with the symptom scores (r = −0·07; P = 0·82).
Fig. 1.
Nasal mucosal T and B lymphocytes during acute cold (a,c) and at the convalescent phase (b,d) in a representative subject. Paraffin sections of nasal mucosal biopsies were stained for CD3 (a,b) and CD20 (c,d). Positive cells are indicated by arrowheads in the epithelium and by arrows in the lamina propria. Scale bars indicate a length of 25 µm.
Fig. 2.
Difference in cell counts (median and interquartile range) between the acute and convalescent phases of natural colds (acute = acute infection, conv = convalescent phase 21 days later, CD3 = T lymphocytes, CD20 = B lymphocytes).
Table 2.
Nasal epithelial mononuclear inflammatory cells/mm2 (median and ranges) in 22 adults having natural colds
| Mast cells | T lymphocytes | B lymphocytes | Macrophages | |||||
|---|---|---|---|---|---|---|---|---|
| Microbiological findings | Acute | Conv | Acute | Conv | Acute | Conv | Acute | Conv |
| All subjects(n= 22) | 18 (0 196) | 0 (0 107) | 474 (114 1714) | 102 (0 487)† | 0 (0 82) | 0 (0 27) | 33 (0 131) | 21 (0 207) |
| No virus found (n = 8) | 55 (0 129) | 32 (0 107) | 400 (114 457) | 81 (0 107) | 3 (0 14) | 0 (0 27) | 18 (0 46) | 21 (0 107) |
| Virus found (n = 14)* | 0 (0 196) | 0 (0 56) | 759 (450 1714) | 115 (0 487) | 0 (0 82) | 0 (0 0) | 37 (0 131) | 30 (0 207) |
| ″Rhino (n = 6) | 0 | 0 | 614 | 39 | 0 | 0 | –‡ | 42 |
| ″Adeno (n = 4) | 29 | 0 | 800 | 124 | 0 | 0 | 33 | 18 |
| ″Parainfluenza | 83 | 28 | –‡ | 122 | 10 | 0 | 21 | 134 |
| ″1 and 3 (n = 2) | ||||||||
| No nasopharyngeal | 36 (0 166) | 0 (0 107) | 400 (114 614) | 106 (0 487) | 0 (0 20) | 0 (0 7) | 29 (0 46) | 31 (0 207) |
| pathogen (n = 12) | ||||||||
| Nasopharyngeal | 0 (0 196) | 36 (0 74) | 759 (4361 714) | 66 (0 160) | 0 (0 82) | 0 (0 27) | 33 (0 131) | 0 (0 107) |
| pathogen (n = 10) | ||||||||
Acute = acute infection, Conv = convalescent phase 21 days later.
Data on one subject who had enterovirus and one with both rhino and respiratory syncytial virus are not shown in the results concerning specific viruses.
Difference between acute and convalescent phases; P = 0·01 (Wilcoxon ranked-pairs analysis).
Data missing due to the absence of the epithelium.
Intraepithelial T lymphocyte counts were significantly higher in the subjects who had proven viral infection or a finding of pathogenic bacteria in the nasopharynx compared to the subjects without such findings (P = 0·005 and P = 0·04, respectively) (Table 2).
T lymphocytes were the most common cells in the lamina propria as well, but their number was smaller than in the epithelium (Fig. 1, Table 3). The numbers of T lymphocytes, B lymphocytes and mast cells were significantly higher in the acute phase than in the convalescent phase (Fig. 2, Table 3). The cell count findings in the acute phase were not associated with the viral findings (P = 0·32 for T lymphocytes, P = 0·24 for B lymphocytes and P = 0·12 for mast cells). The cell counts did not correlate significantly with the symptom scores (r = −0·06, P = 0·81 for T lymphocytes; r = −0·10, P = 0·70 for B lymphocytes and r = 0·01, P = 0·98 for mast cells).
Table 3.
Nasal subepithelial mononuclear inflammatory cells/mm2 (median and ranges) in 22 adults having natural colds
| Mast cells | T lymphocytes | B lymphocytes | Macrophages | |||||
|---|---|---|---|---|---|---|---|---|
| Microbiological findings | Acute | Conv | Acute | Conv | Acute | Conv | Acute | Conv |
| All subjects (n = 22) | 129 (61 408) | 82 (14 136)† | 232 (31 760) | 102 (5 204)‡ | 112 (5 495) | 51 (0 327)§ | 36 (0 189) | 46 (5 138) |
| No virus found (n = 8) | 142 (95 408) | 82 (54 102) | 240 (31 735) | 92 (5 204) | 127 (5 255) | 99 (5 173) | 26 (1 051) | 41 (5 117) |
| Virus found (n = 14)* | 129 (61 306) | 95 (14 136) | 219 (102 760) | 112 (31 163) | 112 (10 495) | 43 (0 327) | 41 (0 189) | 64 (20 138) |
| ″Rhino (n = 6) | 166 | 82 | 287 | 143 | 41 | 56 | 5 | 36 |
| ″Adeno (n = 4) | 187 | 102 | 218 | 71 | 102 | 15 | 44 | 77 |
| ″Parainfluenza | 78 | 81 | 469 | 112 | 137 | 43 | 67 | 99 |
| ″1 and 3 (n = 2) | ||||||||
| No nasopharyngeal | 122 (61 1224) | 64 (14 136) | 229 (31 735) | 112 (71 204) | 112 (5 177) | 43 (5 184) | 32 (0 102) | 46 (20 122) |
| pathogen (n = 12) | ||||||||
| Nasopharyngeal | 177 (82 408) | 88 (82 102) | 248 (143 760) | 51 (5 143) | 127 (36 495) | 125 (0 327) | 41 (26 189) | 53 (5 138) |
| pathogen (n = 10) | ||||||||
Acute = acute infection, Conv = convalescent phase 21 days later.
Data on one subject who had enterovirus and one with both rhino and respiratory syncytial virus are not shown in the results concerning specific viruses.
Difference between acute and convalescent phases; P = 0·006 (Wilcoxon ranked-pairs analysis).
Difference between acute and convalescent phases; P = 0·01 (Wilcoxon ranked-pairs analysis).
Difference between acute and convalescent phases; P = 0·03 (Wilcoxon ranked-pairs analysis).
The finding of pathogenic nasopharyngeal bacteria did not associate with any of the cell counts in the lamina propria in the acute phase (Table 3). The peripheral blood lymphocyte counts during the acute phase had a significant negative correlation with the subepithelial B lymphocyte counts (r = − 0·50, P = 0·04), but not with the T lymphocyte counts (r = − 0·31, P = 0·24).
DISCUSSION
We found more intraepithelial T lymphocytes and subepithelial T and B lymphocytes and mast cells in the nasal mucosa of adult volunteers during the acute stage of naturally acquired common colds compared to the convalescent phase. The increase in subepithelial B lymphocyte counts associated with decrease in peripheral blood lymphocyte counts. These findings indicate that a viral respiratory infection causes T and B lymphocyte and mast cell responses in the nasal mucosa. Although the number of cases was small and only a few different viruses were identified, it seems that the cellular responses were similar irrespective of the viral aetiology of the infection, with the exception that intraepithelial T lymphocyte counts were higher in the subjects with a proven viral infection than in those without. This finding is in accordance with the previous works, which have shown increased T lymphocyte counts in nasal secretions and a decreased number of circulating lymphocytes during experimental rhinovirus colds [5,16]. Moreover, it has been reported recently that the levels of several proinflammatory cytokines, IL-1β, IL-6 and RANTES, which are potent lymphocyte activators, are increased in nasal lavage during natural colds [7]. Based on the findings of experimental rhinovirus infections, however, it has been claimed that the number of mucosal lymphocytes remains unaltered [6,17]. It may be that experimental viral infection results in such mild inflammation that the kinds of immune response seen in symptomatic natural colds are lacking.
Respiratory viruses potentiate leucocyte histamine release [18,19]. Intranasally administered nedocromil and sodium cromoglycate, which are potent mast cell stabilizers, reduce the severity of natural upper respiratory tract infections [20,21]. In accordance with these observations we found increased numbers of mast cells in the subepithelial layer of the nasal mucosa. On the other hand, however, unaltered mast cell numbers in biopsy samples taken from the nasal mucosa [6] and unchanged histamine levels in nasal secretions [4] have been reported during experimental rhinovirus infections.
We observed that the colonization of the nasopharynx by pathogenic bacteria enhanced the intraepithelial T and B lymphocyte response in the nasal mucosa. This is in agreement with the observation that the bacteria and the epithelial cells have interactions that stimulate the release of inflammatory mediators, such as cytokines and chemokines, increasing the severity of the respiratory inflammation [22].
We used a prospective case series design and studied natural colds instead of experimentally induced viral colds. Although this meant that true baseline levels could not be obtained, as subjects were recruited on the development of symptoms it was, however, important because previous work has suggested differences between experimental and natural respiratory infections, with experimentally induced infections producing fewer symptoms [8]. Results from the questionnaire revealed that all volunteers had symptoms compatible with a respiratory infection that either disappeared or lessened during follow-up. We were able to verify the viral aetiology in 64% of the cases, which is similar to other studies dealing with natural colds [7]. There were no significant differences in the clinical characteristics or symptoms scores between the subjects with and without viruses identified. The subjects were not recruited during the period of seasonal allergies (from the end of May to the beginning of August), to avoid allergy symptoms that could have confounded the cold symptoms. The epithelium was missing in 23% of the subjects during the infection and in 17% during the convalescent phase, but this was not related to the viral aetiology, the finding of pathogenic bacteria or the symptoms. Moreover, the subepithelial cellular response was similar irrespective of the presence of epithelium. Thus, we believe that the epithelial detachment most probably represents a technical artefact.
In summary, we found increased numbers of intraepithelial T lymphocytes and subepithelial T and B lymphocytes and mast cells in the nasal mucosa during early natural colds compared to the convalescence phase, indicating T and B cell activation. Moreover, we found that the presence of pathogenic bacteria in the nasopharynx amplified the intraepithelial T cell responses. These findings differ from the observations on experimental virus infections but are supported by other studies carried out during natural viral colds.
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