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Basophil testing with CD63 in pollen-sensitized patients is independent of the circadian clock

2018; Elsevier BV; Volume: 141; Issue: 5 Linguagem: Inglês

10.1016/j.jaci.2017.10.048

1097-6825

Cecilie Lind, Søren Helbo Skaarup, Axel Lorentz, Hans Jürgen Hoffmann,

Food Allergy and Anaphylaxis Research

Allergy is a public health issue that affects more than 25% of western populations. Diurnal variation in allergic symptoms is well documented; exacerbations frequently occur at night and in the early morning, and may be associated with a diurnal lowering of the threshold for histamine sensitivity in the bronchial tree.1de Vries Goei J.T. Booy-Noord H. Orie N.G. Changes during 24 hours in the lung function and histamine hyperreactivity of the bronchial tree in asthmatic and bronchitic patients.Int Arch Allergy Appl Immunol. 1962; 20: 93-101Crossref PubMed Scopus (179) Google Scholar The pathomechanism underlying these observations is not understood, but is thought to involve mast cell mediators. Diurnal variation of clock genes and release of histamine and synthesis of cysteinyl leukotrienes were measured from human intestinal mast cells synchronized with dexamethasone.2Baumann A. Gonnenwein S. Bischoff S.C. Sherman H. Chapnik N. Froy O. et al.The circadian clock is functional in eosinophils and mast cells.Immunology. 2013; 140: 465-474Crossref PubMed Scopus (56) Google Scholar Basophil activation test (BAT) is a useful second-line test for allergy that documents cross-linking of IgE by allergen3Hoffmann H.J. Santos A.F. Mayorga C. Nopp A. Eberlein B. Ferrer M. et al.The clinical utility of basophil activation testing in diagnosis and monitoring of allergic disease.Allergy. 2015; 70: 1393-1405Crossref PubMed Scopus (258) Google Scholar and can be used to document and monitor the severity of allergy. Basophil granulocytes are short-lived blood leukocytes, whereas mast cells mature, and live in tissue for up to 3 months. Although basophil sensitivity has been shown to be very reproducible over a 2-week interval,4Glaumann S. Nopp A. Johansson S.G.O. Borres M.P. Nilsson C. Oral peanut challenge identifies an allergy but the peanut allergen threshold sensitivity is not reproducible.PLoS One. 2013; 8: e53465Crossref PubMed Scopus (56) Google Scholar evidence of diurnal variation on basophil testing would severely limit this useful technique. A recent study identified variation in expression of mRNA for clock genes Per1 and Per2 in human basophils, suggesting circadian influence on the IgE-mediated basophil response.5Ando N. Nakamura Y. Ishimaru K. Ogawa H. Okumura K. Shimada S. et al.Allergen-specific basophil reactivity exhibits daily variations in seasonal allergic rhinitis.Allergy. 2015; 70: 319-322Crossref PubMed Scopus (29) Google Scholar Functional data suggest that these genes may influence basophil activity; surface expression of the activation marker CD203c was higher at 6.00 h than at 18.00 h.5Ando N. Nakamura Y. Ishimaru K. Ogawa H. Okumura K. Shimada S. et al.Allergen-specific basophil reactivity exhibits daily variations in seasonal allergic rhinitis.Allergy. 2015; 70: 319-322Crossref PubMed Scopus (29) Google Scholar We could not confirm this observation in a pilot study,6Baumann A. Skjold T. Hoffmann H.J. Lorentz A. Diurnal variation of CD63 expression on activated blood basophils: a pilot study.Ann Allergy Asthma Immunol. 2016; 116: 77-78Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar in which neither reactivity (% CD63+ basophils) nor sensitivity (the allergen concentration leading to half maximal activation of basophils [EC50] or CD-sens: 100× the inverse of EC50) of basophils of nonallergic individuals stimulated with anti-FcεRI antibody was subject to diurnal variation. Here, we assessed the presence of diurnal variation in basophil reactivity and sensitivity to activation by clinically relevant allergen. We investigated 11 allergic young adults (median age, 26 years) over a 24-hour period outside the pollen season. Volunteers completed an allergy questionnaire of the Allergy Center at Aarhus University Hospital and were included on clinical symptoms of seasonal rhinoconjunctivitis and specific IgE to either grass (n = 9) or birch (n = 2). For clinical characteristics, see Table I. Peakflow, BAT, and serum melatonin measurement were performed at 6 time points (13.00 h, 16.00 h, 19.00 h, 1.00 h, 4.00 h, and 7.00 h) as previously described.6Baumann A. Skjold T. Hoffmann H.J. Lorentz A. Diurnal variation of CD63 expression on activated blood basophils: a pilot study.Ann Allergy Asthma Immunol. 2016; 116: 77-78Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar Basophils were identified with CD193 and activation assessed by incubation at 37°C for 30 minutes with the most relevant allergen, based on the patient history and specific IgE, in nine 10-fold dilutions starting with 1000 specific quality units (SQU)/mL ALK Aquagen and markers CD63 and CD203c (Biolegend, San Diego, Calif). Statistical analyses were performed with Prism 6.0 (GraphPad, La Jolla, Calif), and the dose/response was fitted to a 4-parameter nonlinear regression model. Maximal activation, basophil reactivity, and basophil sensitivity EC50 were calculated. P value of more than .05 in a 1-way ANOVA excluded diurnal variation. In a paired t test, used to test for differences at 12-hour interval, P value of less than .05 was considered significant. Retrospective diurnal allergic symptoms of the volunteers varied from every night to few times a week in 7 of 11 participants (Table II). Three of 7 reported that the frequency of nocturnal symptoms increased in the pollen season. Peak flow measurements confirmed diurnal variation in lung function (P = .0033) (Fig 1, A). Serum melatonin levels varied significantly over 24 hours (P < .0001), confirming a functional circadian clock in the volunteers (Fig 1, B). We found that the fraction of CD63-positive basophils response did not vary over a 24-hour period (Fig 1, C), but a significant difference in the fraction of CD203c-positive basophils with 1-way ANOVA (P = .034) reflected an increase in CD203c reactivity at low allergen concentration between 13.00 H and 1.00 H (P = .0431) consistent with diurnal variation in CD203c expression (Fig 1, D). However, when we compared basophil reactivity and sensitivity for CD63 and CD203c, there was no significant variation (Fig 1, E and F). There was no diurnal variation in the resting mean fluorescence intensity for CD63 or CD203c on blood basophils (not shown). We previously found a significant difference in mean fluorescence intensity6Baumann A. Skjold T. Hoffmann H.J. Lorentz A. Diurnal variation of CD63 expression on activated blood basophils: a pilot study.Ann Allergy Asthma Immunol. 2016; 116: 77-78Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar; that could not be confirmed in this study with allergic volunteers.Table IClinical characteristicsSex: female/male8/3Age (y), median (range)26 (20-46)Duration of allergy (y), median17.64Atopy in family8 of 11Monosensitized1 of 11Grass specific (g6) IgE (KUA/L) (N = 9), median47.31Birch specific (t3) IgE (KUA/L) (N = 2), median1.65Other sensitizations (N) House dust mite (d1)7 of 11 Birch (t3)5 of 11 Mugwort (w6)8 of 11 Dog (e5)7 of 11 Cat (e1)6 of 11Seminal clinical characteristics of patients enrolled in this study are presented as either fraction of patients presenting with a condition, or median of a metric. Open table in a new tab Table IISelf-reported frequency of nocturnal symptoms (“do you have nighttime symptoms from the lung or nose”)Subject no.JanFebMarAprMayJuneJulyAugSepOctNovDec1∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.2∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.3†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.4∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.‡Every night.‡Every night.‡Every night.‡Every night.‡Every night.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.5†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.6‡Every night.‡Every night.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.‡Every night.‡Every night.‡Every night.‡Every night.‡Every night.‡Every night.‡Every night.7‡Every night.‡Every night.‡Every night.‡Every night.‡Every night.‡Every night.‡Every night.‡Every night.‡Every night.‡Every night.‡Every night.‡Every night.8∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.9∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.10∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.†More than 1 time a week.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.11∗Less than 2 times a month.∗Less than 2 times a month.§Less than 1 time a week.§Less than 1 time a week.∗Less than 2 times a month.∗Less than 2 times a month.∗Less than 2 times a month.§Less than 1 time a week.§Less than 1 time a week.§Less than 1 time a week.∗Less than 2 times a month.∗Less than 2 times a month.∗ Less than 2 times a month.† More than 1 time a week.‡ Every night.§ Less than 1 time a week. Open table in a new tab Seminal clinical characteristics of patients enrolled in this study are presented as either fraction of patients presenting with a condition, or median of a metric. In this study, we found expression of CD63 to be a stable marker for allergen activation of basophil granulocytes that did not vary over the investigated 24-hour period. We did see the expected significant variation in plasma melatonin concentration, peak expiratory flow (PEF) and subjective symptoms. The well-known variation in allergic symptoms is thus not reflected in upregulation of CD63 to the cell surface, a common marker for basophil degranulation. We thus strengthen our previous finding, that CD63 expression is not subject to diurnal variation, now with relevant allergen rather than with anti-FcεRI antibody and at 6 time points to allow us to perform relevant statistical analyses.6Baumann A. Skjold T. Hoffmann H.J. Lorentz A. Diurnal variation of CD63 expression on activated blood basophils: a pilot study.Ann Allergy Asthma Immunol. 2016; 116: 77-78Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar Upregulation of CD203c on basophils was significant at low allergen concentration as well as in analysis of diurnal variation, but did not achieve statistical significance in basophil reactivity and sensitivity. Both CD63 and CD203c are validated as a marker for basophil activation.3Hoffmann H.J. Santos A.F. Mayorga C. Nopp A. Eberlein B. Ferrer M. et al.The clinical utility of basophil activation testing in diagnosis and monitoring of allergic disease.Allergy. 2015; 70: 1393-1405Crossref PubMed Scopus (258) Google Scholar Where CD63 is located in the secretory lysozymes containing preformed mediators such as histamine and is truly associated with anaphylactic degranulation of blood basophils, CD203c is found in small vesicles.7MacGlashan D. Expression of CD203c and CD63 in human basophils: relationship to differential regulation of piecemeal and anaphylactic degranulation processes.Clin Exp Allergy. 2010; 40: 1365-1377Crossref PubMed Scopus (139) Google Scholar Our and others4Glaumann S. Nopp A. Johansson S.G.O. Borres M.P. Nilsson C. Oral peanut challenge identifies an allergy but the peanut allergen threshold sensitivity is not reproducible.PLoS One. 2013; 8: e53465Crossref PubMed Scopus (56) Google Scholar data suggest that there is little variation in BAT through CD63. Ando et al5Ando N. Nakamura Y. Ishimaru K. Ogawa H. Okumura K. Shimada S. et al.Allergen-specific basophil reactivity exhibits daily variations in seasonal allergic rhinitis.Allergy. 2015; 70: 319-322Crossref PubMed Scopus (29) Google Scholar and we show that CD203c may be subject to diurnal variation, but an association with circadian rhythm must still be ascertained. Other explanations for diurnal variation in allergic symptoms may be sleeping posture, environmental factors,8Litinski M. Scheer F.A. Shea S.A. Influence of the circadian system on disease severity.Sleep Med Clin. 2009; 4: 143-163Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar or a circadian tissue response.1de Vries Goei J.T. Booy-Noord H. Orie N.G. Changes during 24 hours in the lung function and histamine hyperreactivity of the bronchial tree in asthmatic and bronchitic patients.Int Arch Allergy Appl Immunol. 1962; 20: 93-101Crossref PubMed Scopus (179) Google Scholar Mast cells express clock genes2Baumann A. Gonnenwein S. Bischoff S.C. Sherman H. Chapnik N. Froy O. et al.The circadian clock is functional in eosinophils and mast cells.Immunology. 2013; 140: 465-474Crossref PubMed Scopus (56) Google Scholar and IgE-mediated release of tryptase from dexamethasone-synchronized mast cells in vitro displays diurnal variation,2Baumann A. Gonnenwein S. Bischoff S.C. Sherman H. Chapnik N. Froy O. et al.The circadian clock is functional in eosinophils and mast cells.Immunology. 2013; 140: 465-474Crossref PubMed Scopus (56) Google Scholar indicating that the mast cell response could contribute to diurnal allergic symptoms. However, diurnal variation in skin reactivity to allergen and histamine was similar,9Vichyanond P. Nelson H.S. Circadian variation of skin reactivity and allergy skin tests.J Allergy Clin Immunol. 1989; 83: 1101-1106Abstract Full Text PDF PubMed Scopus (23) Google Scholar suggesting that the variation resides in the tissue response to histamine rather than in mast cell reactivity.9Vichyanond P. Nelson H.S. Circadian variation of skin reactivity and allergy skin tests.J Allergy Clin Immunol. 1989; 83: 1101-1106Abstract Full Text PDF PubMed Scopus (23) Google Scholar We see little evidence for a role of the basophil granulocyte in this mechanism. In conclusion, CD63 is a reliable marker for ex vivo allergen activation of basophil granulocytes. It was stable during a 24-hour period and is therefore the first choice of basophil activation marker.