key: cord-0823047-o6y1rocd authors: CALLOW, KATHLEEN A.; TYRRELL, D. A. J.; SHAW, R J; FITZHARRIS, PENNY; WARDLAW, A. J.; KAY, A. B. title: Influence of atopy on the clinical manifestations of coronavirus infection in adult volunteers date: 2006-04-27 journal: Clin Exp Allergy DOI: 10.1111/j.1365-2222.1988.tb02851.x sha: 9b21ef3b53ab2efe26c216a605979f670d4129b5 doc_id: 823047 cord_uid: o6y1rocd In an attempt to understand the relationship between viral upper respiratory tract infection and the underlying virological and immunological mechanisms, thirty‐four volunteers were inoculated intranasally with coronavirus 229E; subsequent virus shedding and/or antibody rises, indicating active infection, were observed in twenty‐nine. There was a greater increase in independently measured scores of clinical severity, e.g. cold symptoms, in those with detectable IgE in nasal secretions (P < 0.01). A similar association was found between clinical scores and serum IgE concentrations 150 IU/ml, but the relationship with systemic atopy, as assessed by skin‐prick tests to common allergens, was less marked. A more detailed study of twelve of the infected volunteers failed to explain these findings on the basis of mast ceil mediator release, as concentrations of leukotriene B(4), the sulphidopeptide leukotriene C(4), and histamine, were not appreciably elevated in the nasal secretions following virus inoculation. Similarly, there was no evidence that circulating coronavirus specific IgE was produced. Thus, this study suggests that atopy may be related to the severity of cold symptoms produced by coronavirus 229E, although the exact connection has yet to be determined. In allergic subjects, particularly asthmatics, exacerbations commonly occur during respiratory virus infections suggesting the possible involvement of IgE [1] . There is little experimental evidence to confirm the relationship between symptoms of viral infection and atopy, despite the ready availability of measures of atopy, such as reactions to skin-prick testing and circulating IgE concentration [2] [3] [4] [5] . Local IgE in secretions may also be important, as it is also associated with atopy [6, 7] and may be elevated even when serum concentrations are normal [S] . In this study all these measures of atopy have been made in volunteers infected with coronavirus 229E and compared with the severity ofthe cold, assessed subjectively and objectively by an independent medical observer. In order to elucidate any direct role of mast cells or other mediator releasing cells, measuremetit was made of recognized mediators of nasal allergy in twelve of these infected volunteers. Both the sulphidopeptide leukotriene C4 (LTC4) and leukotriene B4 (LTB4), as well as histamine. have been shown to be released into nasal secretions after nasal allergen challenge [9.10] . Histamine also appears to be released into nasopharyngeal secretions during parainfluenza and respiratory syncytial virus (RSV) infections [11. 12] . In parainfluenza. RSV and rubella infections, specific serum and nasal IgE concentrations have been shown to be raised [11] [12] [13] and it has been suggested that it contributes to the wheezing in respiratory infections [11, 12] . Finally, evidence of increased infiltration ofthe nasal mucosa by leucocytes during infection was also sought. Normal adult volunteers, between the ages of 18 years and 50 years, were recruited by a procedure that excludes those with recognized allergic disease, e.g. allergic rhinitis. Isolation, inoculation and assessment procedures have been described elsewhere [14. 15] . Thirty-four volunteers, twenty-ftve females and nine males, were inoculated intranasally with coronavirus 229E, contained in a filtered nasal wash, as previously described [16] . The trial was approved by the Northwick Park Ethical Committee. A daily clinical score was calculated from the record of nasal blockage, rhinorrhoea, sneezing and systemic symptoms. The numbers of used tissues and pyrexia are given heavy weighting [17] . The clinical score of all volunteers before virus challenge was zero. These were performed daily on 3 consecutive days before, and for 6 days after, virus inoculation. After volunteers refrained from nasal blowing for 1 hr. I-ml portions of normal saline were instilled into each nostril with the head tilted backwards 30 . The individual expelled the mixture of mucus and saline into a plastic beaker [10] . This was repeated until 8 ml of wash fluid were recovered, followed by vortex mixing with glass beads to disperse the mucus. Aliquots for immunoglobuhn measurement were centrifuged to sediment the mucus, and supernatants were stored at -20 C. Other aliquots for histamine and leukotriene assay, and virus culture were stored at -70 C. The presence of virus was detected using the C-16 line of MRC-C cells [18] . Enzyme-linked immunosorbent as.say (ELISA) for total IgE in nasal wa.shings Washing methods, substrate and optical density {OD) measurements have been described elsewhere [16] . Nunc round-bottomed immunoplates (Gibco Ltd, Uxbridge, U.K.) were coated with rabbit anti-human IgE antiserum (Hoechst UK Ltd. Hounslow. U.K.) at 1:3000 overnight at 4 C. After washing, plates were incubated with 01% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 2 hrat room temperature (RT). Nasal washings were added, undiluted, and incubated for 6 hr at 4 C. Goat anti-human IgE conjugated to alkaline phosphatase (Sigma Chemical Co., Ltd. Poole. U.K.) at 1:1000 was then added and left overnight at 4 C. After addition of the substrate, plates were read at 2 hr with a Titertek Multiskan ELISA reader (Flow Laboratories Ltd, Irvine. U.K.). Washings were considered positive for IgEiftheOD was ^OD-}-2 standard deviations (s.d.) of that of the controls without nasal washing. This represented a minimum detectable limit of about 0-2 IU/ml. IgE in sera collected prior to. 6 days (intermediate), and 21 days (convalescent) after virus challenge, was measured as described above, omitting the blocking step with BSA. A standard curve on each plate used a standard human serum IgE (provided by Dr E.B. Mitchell, fortnerly ofthe Clinical Research Centre. Harrow, U.K.), and serum samples were diluted at I and .', in 0-1 % BSA and incubated for 4 hr at RT. The OD of the dilution that fell on the linear part ofthe standard curve was used to calculate the concentration. Concentrations of IgE > 150 IU/ml were considered to be abnormally high [4, 19, 20] . Nasal wash fluid was mixed with an equal volume of HPLC grade methanol (Rathburn, Walkerburn. U.K.) using a Silverson grinder (Silverson Ltd, Chesham, U.K.) and stored at -20 C. After acidification to pH 40 with acetic acid the precipitate was removed by centrifugation (12000x^. 10 min. 4 C) and the supernatant was diluted to give a lO'/n methanol H:O solution. The leukotrienes were then extracted using C18 Sep-paks (Water Associates, Northwick, U.K.) [10] and resuspended in PBS buffer. The measurement of LTC4 and LTB4 by a double antibody radioimmunoassay (using antisera supplied by Dr E. Hayes. Merck Sharp & Dohme, Rahway, NJ, U.S.A.) has been described in detail elsewhere [21] [22] [23] . Briefly, rabbit anti-LTC4 or anti-LTB4 were incubated with unknown or dilutions of synthetic LTC4 or LTB4 (a gift from Dr J. Rockach, Merck Forest Laboratories. Quebec, Canada) together with 14.I5-'H-LTC4 or 14,15-^H-LTB4 (New England Nuclear (NEN), Southampton. U.K.) together with normal rabbit immunoglobulins in PBS containing p-methylsulphonyl fluoride (I x 10 *'M)andO 25% gelatin. After 2hr incubation at 4^, goat anti-rabbit immunoglobulin (Biorad, Watford, U.K.) diluted in 5% polyethylene glycol (PEG) 6000 was added. Two hours later the tubes were centrifuged (15 000 x;?. 15 min. 4 C). The radioactivity in the supernatant was measured with an LKB beta-1217 counter using Aquasol II (NEN) as scintillant. All assays were performed in duplicate and the leukotriene concentration determined using a smoothed spline computed analysis. Recovery experiments using synthetic leukotrienes confirmed a recovery of 82-1-9% for LTC4 and 100% for LTB4. The baseline pre-challenge nasal washing leukotriene concentration was obtained from the mean of 3 consecutive days prior to virus inoculation. Recovery and measurement of histamine from na.sal washings After collection ofthe nasal washing, an aliquot of 200 //I wash fluid was mixed with 20 }i\ of 0-1 M EDTA pH 7-4 and stored at -80 C. Histamine measurement used a specific isotope enzymatic assay modified from that of Guilloux et al. [24] . which had a sensitivity of 05 ng/mi. In brief, the sample was incubated with methyl transferase obtained from rat kidney in the presence ofthe tritium-labelled methyl donor s-adenylmethionine. The labelled methyl histamine so formed was extracted in chloroform and the activity of the sample compared with a standard curve obtained using histamine dihydrochloride (Sigma Ltd). On one set of samples a fluorimetric assay for histamine was performed and the results agreed closely with those obtained using the above methods. Indirect ELISA. Plates were coated with coronavirus 229E or control antigen as described elsewhere [16] . Sera diluted I in BSA diluent were incubated for 5 hr at RT, and IgE was detected as described for total IgE. The peroxidase-conjugated goat antihuman IgE (Miles Laboratories Ltd, U.K.), used by Welliver et al. [11, 12, 25] , was not used as it was found to have a low-level cross-reactivity with IgG. IgE capture ELISA. Plates were coated with anti-human IgE produced in goats (Hoechst) overnight as described for measurement of total IgE. Sera were diluted I or ro in BSA diluent and incubated for 3 hr at RT. Virus or control antigen at 5% was added and left for 3 hr at RT. Rabbit antiserum to coronavirus 229E diluted 1:1000 was added and left overnight at 4 C. followed by alkaline phosphatase conjugated to antirabbit IgG (Miles Laboratories) at 1:4000 for 4 hr al RT. Substrate was added and ODs read at 405 nm as described. Using both methods for measuring specific IgE, optimal dilutions of reagents were determined by chequerboard titrations. Optical densities obtained with control antigens were subtracted from those obtained with virus. Sera were considered positive if these adjusted ODs were >0D + 2 s.d, ofthe controls without serum. Skin-prick tests were performed using standard allergens to grass pollen, trees, shrubs, Dermatophagoides sp., house dust, cat, dog, negative and positive histamine control (Pharmacia, Uppsala, Sweden). None ofthe volunteers developed a weal ^3 mm to the negative control. Nasal cytology smears were collected before and 3 and 6 days after virus challenge, by gently scraping the interior turbinate of each nostril with a specially designed nasal probe. The material was smeared onto a glass microscope slide, allowed to air-dry, fixed in methanol and stained with May-Grunwald/Giemsa. The slides were coded and counted blind by two independent investigators. A differential count was performed counting 200 cells. The statistical analyses were performed on a Sirius I microcomputer using the program. Statistical Package for Personal Computers (SPP, Patrick Royston, Clinical Table I Research Centre, Northwick Park Hospital. Harrow, U.K.)-For correlating scores with IgE or leukotriene concentrations. Spearman's coefficient of rank correlation was calculated. The other non-parametric test used was a rank analysis of variance. Of thirty-four volunteers inoculated with coronavirus 229E. Iwenty-nine became infected as indicated by virus shedding and twenty-three had some respiratory symptoms, with clinical scores ranging from 0-5 to 71-5 (Table I) same individuals, had high (^150 IU/ml) serum IgE concentrations before the trial (Table I) . Seventeen volunteers had detectable amounts of nasal IgE and only eight were negative for all three measures of atopy. Those volunteers who had detectable nasal IgE had almost four times as much serum IgE compared with those who had no detectable IgE (Fig. 1) . However, eleven volunteers who had negative skin tests had detectable nasal IgE and three had high concentrations of IgE in their sera ( Table 1) . None of these relationships was significant at P<005. The presence or amount of local and serum IgE and the result of skin tests were all related to clinical scores (Fig. 2) . The mean clinical score of those with detectable amounts of nasal IgE was over three times higher than the mean score of those with undetectable IgE{/'<001). The mean clinical scoreof those with higher than normal serum IgE was about one and a half times higher than that of those volunteers whose serum IgE was within the normal range, although the difference was not statistically significant (in a two-tailed test). The mean clinical score of volunteers with positive skin tests was slightly higher than those with a negative test, although again this did not reachstatisticalsignificance. Groups that were positive for all three measures of atopy, or at least two of them, had a considerably higher mean score than the rest, although the differences were not statistically significant. The group that was positive for at least one of the criteria had a mean score of 23-8 compared with 4-7 for those who were negative for all three (/'-OOOl). None of ihe indicators of atopy appeared to be related to the amount of duration of virus shedding or to the size of specific IgG antibody rises (data not shown). The following studies were carried out in twelve ofthe infected volunteers lo try to elucidate the role of possible mediators. The concentralioti of the leukotrienes LTB4 and LTC4 in nasal washings was very low throughout the trial, compared with concentrations observed following provocation [10] (Fig. 3a) . The mean concentrations showed fluctttations on different days, which did not appear to correlate with the changes in mean clinical scores (Fig. 3b) . However, on most days after infection. LTC4 concentrations in individual volunteers correlated positively with the clinical score, while with LTB4 the reverse was the case; these correlations were not statistically significant however (data not shown). Those volunteers classified as atopic by detectable IgE levels in nasal washing tended to have more LTQ (/* = OO28) and less LTB4 (not significant) in their preinoculation washings than the non-atopies (Table 2 ). These differences persisted throughout the trial but values were so low that they may not be biologically important. Histamine concentrations were also very low or undetectable. Only four volunteers had detectable amounts in their pre-trial washings; all four also had detectable nasal IgF and higher than average LTC4 concentrations ( Table 2 ). There was no indication that histamine concentrations increased after infection (data not shown) while one volunteer with high serum IgE (B.W.) and three with nasal IgE had no detectable histamine at any stage. Optical densities significantly higher than the control were detected in only two prechallenge sera (M.B. and L.M.) using the indirect ELISA. Because of these largely negative results an IgE capture ELISA was then Iried. With this method, none ofthe pre-chalienge sera appeared to contain specific IgE. although the convalescent sera of two volunteers (L.M. and S.C".) appeared to contain trace amounts, as did one intermediate serum (K.G.) . No other samples from any volunteer contained detectable specific IgE. There was also no evidence that total IgE concentrations in the sera increased after infection (data nol shown). Most of the cells in the scrapes were epithelial or goblet cells. Neutrophils were occasionally seen but there were no differences between the pre-and post-infective cytology, excepl in one volunteer (H.G.) where neutrophils were present after infection, and another (S.C.) where neutrophils were seen before infection hut not after. No eosinophils were seen in any subject. The variation in the indiees of atopy used in this study is well recognized [20. 26. 27] although, as in other studies, the best agreement was between serum and nasa! IgE concenlrations [28. 29] . Detectable nasal IgE concentrations were most closely associated with a more severe cold, as assessed by a combination of subjective and objective clinical scores (P< 0-01). followed by elevated serum IgE and positive skintest results. This interrelationship between the clinical seventy of rhinitis and the presence of allergic processes is in accordance with the known capacity of virus infection to trigger asthma attacks in susceptible individuals [I. 30 . 31], Rhinovirus infections have been associated with asthma and bronchitis in both children and adults [L 30. 31], but exacerbations only occurred in a minority of asthmatic adults during experimental rhinovirus infection [32] . No consistent association between the severity of eolds and circulating or local IgE concentrations has yet been found in volunteers infected with rhinovirus (Callow, unpublished observation). A number of possible mechanisms underlying the relationship were explored in this study. There was no evidence of coronavirus-specific circulating IgE prior to. or following, viral inoculation, and it may be that atopic individuals do not become sensitized to viral antigens [33] . In this case, direct triggering of cells, such as nasal mast cells or basophils, via specific surface-bound IgE directed against the virus would be unlikely. The lack of Involvement of mast cells or basophils was further suggested by the failure of this study to identify any rise in the eoncentration of histamine. sulphidopeptide leukotriene d or leukotriene B4 following virus inoculation. The small but statistically significant differences in baseline leukotriene concentrations in atopic and non-atopic volunteers were thought to be too small to be biologically important. Similarly, the cytological findings did not identify any influx of inflammatory cells, in particular eosinophils. characteristic of the allergie response. Although a significant increase of total protein is found in nasal secretions, following virus infection [34. 35; Callow, unpublished observations] , other studies have, like ours, failed to detect a similar increase in hisiamine or leukotriene concentrations [36] . The observed increase in concentrations of these mediators following allergen provocation may be due to the fact that inflammation and release of mediators is due to a large amount of allergen given in one dose whereas in virus infections, such effects are due to small amounts of infectious virus that are released over a much longer period. Furthermore, studies with allergen provocation arc usually done wilh overtly atopic subjects [10] . whereas the atopy detected in our volunteers was mild and not accompanied by allergic symptoms. This study has raised the possibility of a relationship between the symptoms of a cold and the presence of alopy, in particular as detected by intranasal IgE concentrations. Studies attempting to dissect the mechanism of this relationship failed to identify a common pathway for viral and allergic rhinitis. Possibly an immunologica! imbalance, either humoral or cell-mediated, which has been shown to be associated with atot)y [33, 37, 38] may independently lead lo both atopy and increased severity of symptoms of virus infection. Nevertheless, the results of this study show that increasing our understanding of any common pathways between virus-and allergen-induced rhinitis may provide insights which would have therapeutic implications for both conditions. Gregg 1. Provtjcalion of airflow limitation by viral infection: implication for treatment IgE concentrations in children with atopic diseases Some factors influencing the serum IgE levels in atopic diseases Age-relaled serum immunoglobulin E levels in healthy subjects and in patients with allergic disease Distribution or IgE in a community population sample: correlations with age, sex and allergen skin test reactivity Deuschl H. lmmunoglobuUns in nasal secretion with special reference to IgE Immunogtobulins in nasal swretions and nasal mucosa in perennial rhinitis Measurement of specific IgE antibodies in nasal secretion-evidence for local production Peptide leukotriene release after antigen challenge to patients sensitive to ragweed Allergen-induced release of sulphidopeptide leukotrienes (SRS-A) and LTB4 in allergic rhinitis The development ofrespiratory syncytial virus-specific IgE and the release of histamine in nasopharyngeal secretions after infection Role of parainfluenza virusspecific IgE in pathogenesis of croup and wheezing subsequent to infection Kinetics of specific IgA. igD. IgE. IgG and IgM antibody responses in rubella The study of antiviral compounds in volunteers Intranasa! inlerferon as protection against experimental respiratory coronavirus infection in volunteers Effect of specific humoral immunity and some non-specific factors on resistance of volunteers to respiratory coronavirus infection Common Cold and Related Diseases Clones of MRC-C cells may be superior to the parent line for the culture of 229E-like strains of human respiratory coronavirus IgE concentrations measured by PRIST in serum of healthy adults and in palients with respiratory allergy Continuing medical education: human IgE Inhibition of leukotriene Ci and B4 generation by human eosinophils and neulrophils with ihe lipoxygenase pathway inhibitors U-60.257 and BW755C Measuring leukotrienes of slow reacting substance of anaphylaxis: development of a specilic radio-immunoassay Preferential generation of leukotriene C4 by human eosinophils Enzymatic isotopic assay for human plasma histamine Respiratory syncytial virus-specilic IgE responses following infection: evidence for a predominantly mucosal response Jacobs D. C orrelation of skin, nasal and inhalation tests with the IgE in ihe serum, nasal fluid and sputum Population difTerences in cutaneous methacholine reactivity and circulating IgE concentrations Presence of) E in nasal washings and sputum from asthmatic patients Local and serum IgE in vasomotor rhinitis Viruses as precipitants of asthmatic attacks in children Rhinovirus and influenza type A infections as precipitants of asthma Exacerbations of asthma in adults during experimental rhinovirus infection Regulation ofthe IgE system: experimental and clinical aspects Protein composition of nasal secretion during respiratory virus infection Changes in IgA and IgG concentrations in nasal secretions prior to the appearance of antibody during viral respiratory infection in man Inflammatory mediators in nasal secretions during induced rhinitis Regulation of IgE synthesis in man An introduction to allergic disease We thank the staff and volunteers ofthe M RC Common Cold Unit for their assistance in this study.