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Reference values of IgG and IgG4 serum levels specific for inhalant allergens in non-atopic children
Revue française d’allergologie 49 (2009) 410–415
Original article
Reference values of IgG and IgG4 serum levels specific for inhalant allergens in non-atopic children Les valeurs de référence d’IgG et niveaux de sérum IgG4 spécifiques pour allergènes inhalants chez des enfants non atopiques M. De Amici a, S. Quaglini b, R. Moratti c, C. Torre a,d, G.L. Marseglia a,d, G. Ciprandi e,* a
Clinica Pediatrica, Foundation IRCCS San Matteo, University of Pavia, Pavia, Italy b Department of Computer Science and Systems, University of Pavia, Pavia, Italy c Department of Biochemistry, Foundation IRCCS San Matteo, University of Pavia, Pavia, Italy d Clinica Pediatrica, University of Catania, Catania, Italy e Department of Internal Medicine, Azienda Ospedaliera Universitaria San Martino, Genoa, Italy Received 2 February 2009; accepted 26 March 2009 Available online 15 May 2009
Abstract Background. – An IgG response to allergens has been well characterized, however few studies have investigated IgG and IgG4 production in normal subjects. Objective. – The aim of this study was to find reference values for six common inhalant allergens in a population of healthy children. Methods. – One hundred and six females and 107 males, from one to 12 years old, were studied assessing the serum levels of specific total IgG and IgG4 for six allergens. Results. – A homogeneous behaviour was not observed, among allergens or among patient subgroups. Some allergens increased, while others decreased from pre-school to elementary school age. In females, this decrease was less evident, while increases were more evident. Differences were found between seasonal and perennial allergens, the latter showing higher values. We also observed differences between temporal trends of IgG and IgG4 in some patient groups. Percentiles of distributions have been provided as reference values, for all subgroups that were significantly different. Conclusion. – Defining an individual profile would be useful for characterising the specific humoral response. # 2009 Elsevier Masson SAS. All rights reserved. Keywords: Allergen-specific IgG4/IgG levels; Inhalant allergens; Normal children
Résumé Une réponse IgG aux allergènes a été bien caractérisée, cependant peu d’études a examiné IgG et la production IgG4 chez des sujets normaux. Le but de cette étude était de déterminer des valeurs de référence pour six allergènes inhalants communs dans une population d’enfants sains. Les valeurs des IgG et Ig4 totales et spécifiques ont été évaluées chez 106 filles et 107 garçons, de un à 12 ans pour six allergènes. Un comportement homogène n’a pas été observe pour certains allergènes et dans certains sous-groupes de patients. Les concentrations ont augmenté pour certains allergènes, tandis que les valeurs diminuaient entre les âges préscolaires à la rentrée en maternelle. Chez les filles, cette diminution était moins nette à l’inverse des augmentations qui étaient évidentes. Des différences ont été relevées entre allergènes saisonniers et perannuels, les derniers montrant des valeurs plus élevées. Nous avons aussi observé des variations dans le temps pour les IgG et IgG4 chez certains patients. Les distributions des IgG et IgG4 en percentiles ont été établies comme valeur de référence et également dans les sous groupes qui montraient des résultats significativement différents. Il apparaît nécessaire de définir les profils individuels pour caractériser la réponse humorale spécifique. # 2009 Elsevier Masson SAS. Tous droits réservés. Mots clés : IgG4/IgG spécifique d’allergène nivelle ; Inhalant des allergènes ; Enfants normaux
* Corresponding author: Semeiotica e Metodologia Medica I, A.O.U. San Martino, Viale benedetto XV 6, Genoa, Italy. Adresses e-mail: [email protected], [email protected] (G. Ciprandi). 1877-0320/$ – see front matter # 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.reval.2009.03.006
M. De Amici et al. / Revue française d’allergologie 49 (2009) 410–415
1. Introduction The development of IgE antibodies to inhalant or food allergens is a distinctive feature of atopic disease. Although specific serum IgG antibodies directed to these allergens have been detected in both atopic and non atopic subjects [1], higher IgG levels are observed in the latter, while increased levels of antigen-specific IgG4 have been described in patients with atopic dermatitis [2]. A major problem exists in interpretation of the clinical significance of these IgG-associated antibodies in the allergic patient because of the ubiquitous presence of IgG antibody levels as well as the lack of appropriate normative reference values for specific allergen IgG responses in the general population which precludes the delineation of a discriminating cut-off value. Several studies report that IgG4 serve as blocking antibodies in the IgE mediated allergen reaction [2]. Indeed, it has been recently demonstrated that IgG production is dependent on the effects of IL-10, a typical cytokine produced by T regulatory cells (Tregs) with immunosuppressive function [3]. Normal subjects therefore may produce IgG4 to common allergens through T regulatory effects to counterbalance the potentially deleterious effects of IgE production. In contrast, since allergic patients show a Treg defect, their uninhibited production of IgG4 results in a Th2 polarization with production of IgE instead of IgG. One of the currently proposed beneficial mechanisms of specific immunotherapy is its capacity to induce and restore normal Treg function, as evidenced by enhanced IL10 and IgG4 production [4]. Thus, the measurement of specicific serum IgG4 is currently proposed as one of the parameters to monitor patients receiving allergen-specific immunotherapy [4]. As in the case of allergen specific IgG, however, there is no descriptive reference profile for the specific IgG4 subtype. Therefore, the purpose of the present study was to establish reference values for six common inhalant allergens in a paediatric population of two age groups (1–3 versus 7–12) testing whether there are significant differences related to gender, different allergen types (seasonal versus perennial). 2. Methods and statistical analysis Two hundred and thirteen healthy children living in Italy, in rural or urban areas were evaluated. The age and gender distributions of the study population included the following: 60 subjects were from one to three years (30 males, 30 females); 153 subjects were from seven to 12 years (77 males, 76 females). The family histories in first-degree relatives and personal histories were negative for allergic diseases (i.e. atopic dermatitis, wheezing/asthma, and rhinoconjunctivitis). All subjects were negative to both the skin prick test and an in vitro IgE screening test for allergy. The sera of all patients were drawn outside of the seasonal pollination period. The study was approved by the institutional review board of the University of Pavia and informed consent was obtained from at least one parent of each patient before blood was drawn.
411
Serum was separated from all blood samples within four hours and stored at –208 C until testing. Determination of allergen-specific IgG4 and total IgG antibodies was performed on all sera against the following six common inhalant allergens: Dermatophagoides pteronyssinus (D1), Dermatophagoides farinae (D2), Phleum pratense (G6), cat dandruff (E1), dog dandruff (E5), and Parietaria judaica (W21) in a single laboratory session (Phadia AB, Uppsala, Sweden) using an IgG RAST FEIA assay described previously [5]. Briefly, the method consists of the following steps. The test serum was added to the ImmunoCAP (a solid phase to which the allergen of interest is covalently bound) and incubated. After washing to eliminate any non-specific IgG4 or IgG, enzyme-labelled anti-IgG4 or IgG was added to form a complex. After another incubation of 150 min, any unbound labelled anti-IgG4 or IgG antibodies were eliminated by washing. The labelled allergen-antibody complex was then incubated with a developing agent and, after stopping the reaction, the fluorescence of the eluate was measured by fluorimetry (FluoroCount); and specific antibody concentrations were expressed in percentage (fluorescence sample 100/fluorescence reference serum). The higher the response, the more specific IgG or IgG4 is present in the specimen. To evaluate the test results, the response for patient samples were compared directly with a reference serum run in parallel. Since children were sampled at school, individual age values were estimated as the median of pre-school or elementary school age. Since IgG and IgG4 for all the allergens were measured using a fluorescence percentage with respect to a unique reference value and in a single laboratory session, two further measurements were calculated to minimize sampling error. 3. Statistical analysis Descriptive statistics (5th, 50th and 95th percentiles and distribution mode) have been used to characterise the sample for each allergen. The non-parametric Wilcoxon test was used to test for differences between groups, identified by gender, sex, and allergen type. The package S-Plus (MathSoft Corp) was used for all analyses. 4. Results The median, 5th and 95th percentiles of IgG and IgG4 for the six allergens are shown in Table 1. Measurement of IgG responses revealed a high variability for house dust and dog dandruff (D1, D2 and E5) with respect to a lower variability for E1, G6, W21. A significant gender difference was observed for D1 ( p = 0.04), E1 ( p = 0.02) and E5 ( p = 0.005). In the measurement of IgG4 responses, G6 appeared to have a lower variability in comparison responses seen to the other allergens. When gender data were evaluated separately, significant differences were observed for D1 ( p = 0.04) and E1 ( p = 0.002). A significant difference ( p < 0.00001) was noted between the IgG average value of the two seasonal (median 28, interquartile range 12.1–57.4) and the four perennial allergens
412
M. De Amici et al. / Revue française d’allergologie 49 (2009) 410–415
Table 1 Specific IgG and IgG4 for the six allergens in the whole sample and for males and females. Median (5, 95th percentile) Specific IgG All D1 D2 E1 E5 G6 W21 a
46.9 38.8 30.3 36.9 20.4 33.4
Specific IgG4 Male
(16.7, 106.8) (15.9, 87.7) (12.4, 60.7) (15.2, 87.1) (9.9, 42.8) (13.5, 67.6)
46.2 37.5 28.1 30.3 20.8 32.6
Female (11.8, 104.4)a (13.1, 88.2) (9.2, 61.5)a (15, 84.4)a (10.3, 41) (10.9, 67)
47.6 40.5 31.3 40.3 20.1 35.8
(22.4, 110.2)a (20.7, 58.8) (17.9, 58.8)a (17, 88.4)a (9.8, 47.4) (15, 71.7)
All 4.6 4.5 8.1 8.6 3.7 4.1
Male (2.7, (2.8, (3.2, (3.8, (2.6, (2.4,
21.1) 19.1) 18.4) 21.4) 11.6) 15.8)
4.3 4.6 6.3 8.5 3.7 4.1
(2.5, (2.8, (2.8, (3.7, (2.5, (2.3,
Female 19.4)a 19.3) 18.4)a 20.8) 12.1) 16.2)
4.7 4.5 8.8 8.7 3.6 4.2
(3.2, (2.9, (3.8, (4.1, (2.7, (2.7,
23)a 17.7) 16.6)a 27.6) 10.7) 13.8)
Significant difference between males and females.
Fig. 1. Temporal trend of specific IgG and IgG4 in males (30 subjects from one to three years and 77 from seven to 12 years) and females (30 subjects from one to three years and 76 from seven to 12 years).
M. De Amici et al. / Revue française d’allergologie 49 (2009) 410–415
(median 38, interquartile range 15.8–79.7), and this difference was also observed when males and females were compared separately. Similarly, a significant difference ( p < 0.00001) was also observed between the IgG4 average value of the two seasonal (median 4.01, interquartile range 2.71–13.51) and the four perennial allergens (median 6.55, interquartile range 3.43– 19.7), and again this difference held when considering males and females separately. Shown in Fig. 1 is a comparison of the temporal trend of specific IgG and IgG4 (medians) in males and females. Although there was no consistent pattern, some allergens increased and others decreased from pre-school to elementary school age. In females, the decrease was less evident while an increase was more evident. The Wilcoxon tests between the two age classes confirmed this observation. For example, the value 0.002d in the first cell of the table indicates that specific IgG for D1 in males decreased significantly with age ( p = 0.002). Another observation was related to IgG4 responses to dog and cat dandruff, which was clearly higher than responses to the other allergens in both genders. In Fig. 1, it is also notable that temporal trends for IgG, mainly for males, are very different
413
from IgG4 (only G6 shows a similar behaviour). In females, only E1 and G6 showed different trends. In the light of the results reported in Fig. 1, it can be argued that different reference values are necessary for the two age classes for: specific IgG D1, D2, E1, E5 and W21 in males; specific IgG E5 and G6 in females and specific IgG4 E1 and E5 in females. Percentiles of these nine allergens that significantly differ in the two age classes are shown in Fig. 2. Moreover, the difference between the distribution of the average value of perennial and seasonal allergens in the whole sample was also investigated. Perennial allergens showed higher values, the IgG distribution mode was approximately 35% (fluorescence value) for perennial, and approximately 25% for seasonal allergens, whereas the IgG4 distribution mode was approximately 5% for perennial, and around 2.5% for seasonal allergens (Fig. 3). 5. Discussion It has been previously demonstrated that allergic and healthy subjects differ not only in the presence/absence of specific IgE, but also in the preferential expression of distinct IgG
Fig. 2. Percentiles of allergens that significantly differ in the two age classes. p-values from Wilcoxon test comparing the allergens in the two age classes. Pedix specifies decreasing (d) or increasing (i) trend.
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M. De Amici et al. / Revue française d’allergologie 49 (2009) 410–415
Fig. 3. Distributions of the average values of perennial and seasonal allergens.
specificities [1-4,6]. Allergic inflammation is a systemic process involving not only the affected allergen exposed tissue but also the body as a whole, with T and B lymphocytes playing a primary role in inflammation [7]. Recent studies have provided a better understanding of the role of IgG and IgG4 during specific immunotherapy [4,8]. Studies have investigated the clinical significance of food allergen-specific IgE/IgG4 [2]. Although food allergy is known to be mediated by specific IgE antibody, the definitive role of IgG4 in food allergy is unclear. Not only allergic patients have an individual profile, but also healthy subjects have precise defined characteristics. In this context, the present work assesses reference values for the healthy paediatric population. The results of the present study show that specific IgG4 and IgG serum levels in healthy children have considerable variability to different degrees with respect to age as well as gender. Variability with age could be due to a more prolonged exposure to perennial allergens with increasing age. Indeed, there is a different pattern for pollens and perennial allergens. This hypothesis seems to be supported by our result that perennial allergens increase more than seasonal allergens with age. However, a weakness of this observation is based on the fact that two evaluated allergens are pollens, such as grasses and Parietaria. Pollen exposure may influence the levels of IgE production. We were aware of this issue and we performed the study outside the pollen season, enrolling subjects in a brief period. Therefore, this bias could be partially reduced.
In any case, this preliminary study provides evidence of a relevant variability in the IgG and IgG4 profile for common allergens in a normal population of children. The relationship of these values with those for allergic children will require future studies to establish whether the findings of our study will have clinical relevance. Consequently, the usefulness of the individual profile in clinical practice will require validation in larger randomized studies. In conclusion, the results of the present study have suggested age and gender-related differences in specific humoral responses to allergens in a normal healthy population of children further studies comparing established references values with values of allergic children will be required to establish the usefulness of this research. Acknowledgments The authors wish to thank Vania Giunta (Dipartimento di Informatica e Sistemistica, Università di Pavia) for data analysis and Laurene Kelly for correction of the English. References [1] Michils A, Vervier I, Choufani G, et al. Relationship between allergic status and specificity of IgG antibody to inhaled allergens: the grass pollen model. Clin Exp Allergy 1999;29:832–9. [2] Noh G, Ahn HS, Cho NY, et al. The clinical significance of food specific IgE/IgG4 in food specific atopic dermatitis. Pediatr Allergy Immunol 2007;18:63–70.
M. De Amici et al. / Revue française d’allergologie 49 (2009) 410–415 [3] Ling EM, Smith T, Nguyen XD, et al. Relation of CD4+CD25+ regulatory T-cell suppression of allergen-driven T-cell activation to atopic status and expression of allergic disease. Lancet 2004;363: 608–15. [4] Passalacqua G, Durham SR. Allergic rhinitis and its impact on asthma: allergen immunotherapy. J Allergy Clin Immunol 2007;119:881–91. [5] Meier P, Muller U. Evaluation of IgG RAST FEIA for the assay of venomspecific IgG antibodies during venom immunotherapy. Int Arch Allergy Immunol 1998;117. 46–51.
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[6] Duchateau J, Michils A, Michel O, et al. Mite allergy is associated with a specific profile of IgG epitopes recognized on antigen p1 of Dermatophagoides pteronyssinus. Clin Exp Allergy 1997;27:296–305. [7] Kallinich T, Beier KC, Wahn U, et al. T-cell co-stimulatory molecules: Their role in allergic immune reactions. Eur Respir J 2007;29:1246–55. [8] Rossi RE, Monasterolo G, Coco G, et al. Evaluation of serum IgG4 antibodies specific to grass pollen allergen components in the follow up of allergic patients undergoing subcutaneous and sublingual immunotherapy. Vaccine 2007;25:957–64.
Original article
Reference values of IgG and IgG4 serum levels specific for inhalant allergens in non-atopic children Les valeurs de référence d’IgG et niveaux de sérum IgG4 spécifiques pour allergènes inhalants chez des enfants non atopiques M. De Amici a, S. Quaglini b, R. Moratti c, C. Torre a,d, G.L. Marseglia a,d, G. Ciprandi e,* a
Clinica Pediatrica, Foundation IRCCS San Matteo, University of Pavia, Pavia, Italy b Department of Computer Science and Systems, University of Pavia, Pavia, Italy c Department of Biochemistry, Foundation IRCCS San Matteo, University of Pavia, Pavia, Italy d Clinica Pediatrica, University of Catania, Catania, Italy e Department of Internal Medicine, Azienda Ospedaliera Universitaria San Martino, Genoa, Italy Received 2 February 2009; accepted 26 March 2009 Available online 15 May 2009
Abstract Background. – An IgG response to allergens has been well characterized, however few studies have investigated IgG and IgG4 production in normal subjects. Objective. – The aim of this study was to find reference values for six common inhalant allergens in a population of healthy children. Methods. – One hundred and six females and 107 males, from one to 12 years old, were studied assessing the serum levels of specific total IgG and IgG4 for six allergens. Results. – A homogeneous behaviour was not observed, among allergens or among patient subgroups. Some allergens increased, while others decreased from pre-school to elementary school age. In females, this decrease was less evident, while increases were more evident. Differences were found between seasonal and perennial allergens, the latter showing higher values. We also observed differences between temporal trends of IgG and IgG4 in some patient groups. Percentiles of distributions have been provided as reference values, for all subgroups that were significantly different. Conclusion. – Defining an individual profile would be useful for characterising the specific humoral response. # 2009 Elsevier Masson SAS. All rights reserved. Keywords: Allergen-specific IgG4/IgG levels; Inhalant allergens; Normal children
Résumé Une réponse IgG aux allergènes a été bien caractérisée, cependant peu d’études a examiné IgG et la production IgG4 chez des sujets normaux. Le but de cette étude était de déterminer des valeurs de référence pour six allergènes inhalants communs dans une population d’enfants sains. Les valeurs des IgG et Ig4 totales et spécifiques ont été évaluées chez 106 filles et 107 garçons, de un à 12 ans pour six allergènes. Un comportement homogène n’a pas été observe pour certains allergènes et dans certains sous-groupes de patients. Les concentrations ont augmenté pour certains allergènes, tandis que les valeurs diminuaient entre les âges préscolaires à la rentrée en maternelle. Chez les filles, cette diminution était moins nette à l’inverse des augmentations qui étaient évidentes. Des différences ont été relevées entre allergènes saisonniers et perannuels, les derniers montrant des valeurs plus élevées. Nous avons aussi observé des variations dans le temps pour les IgG et IgG4 chez certains patients. Les distributions des IgG et IgG4 en percentiles ont été établies comme valeur de référence et également dans les sous groupes qui montraient des résultats significativement différents. Il apparaît nécessaire de définir les profils individuels pour caractériser la réponse humorale spécifique. # 2009 Elsevier Masson SAS. Tous droits réservés. Mots clés : IgG4/IgG spécifique d’allergène nivelle ; Inhalant des allergènes ; Enfants normaux
* Corresponding author: Semeiotica e Metodologia Medica I, A.O.U. San Martino, Viale benedetto XV 6, Genoa, Italy. Adresses e-mail: [email protected], [email protected] (G. Ciprandi). 1877-0320/$ – see front matter # 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.reval.2009.03.006
M. De Amici et al. / Revue française d’allergologie 49 (2009) 410–415
1. Introduction The development of IgE antibodies to inhalant or food allergens is a distinctive feature of atopic disease. Although specific serum IgG antibodies directed to these allergens have been detected in both atopic and non atopic subjects [1], higher IgG levels are observed in the latter, while increased levels of antigen-specific IgG4 have been described in patients with atopic dermatitis [2]. A major problem exists in interpretation of the clinical significance of these IgG-associated antibodies in the allergic patient because of the ubiquitous presence of IgG antibody levels as well as the lack of appropriate normative reference values for specific allergen IgG responses in the general population which precludes the delineation of a discriminating cut-off value. Several studies report that IgG4 serve as blocking antibodies in the IgE mediated allergen reaction [2]. Indeed, it has been recently demonstrated that IgG production is dependent on the effects of IL-10, a typical cytokine produced by T regulatory cells (Tregs) with immunosuppressive function [3]. Normal subjects therefore may produce IgG4 to common allergens through T regulatory effects to counterbalance the potentially deleterious effects of IgE production. In contrast, since allergic patients show a Treg defect, their uninhibited production of IgG4 results in a Th2 polarization with production of IgE instead of IgG. One of the currently proposed beneficial mechanisms of specific immunotherapy is its capacity to induce and restore normal Treg function, as evidenced by enhanced IL10 and IgG4 production [4]. Thus, the measurement of specicific serum IgG4 is currently proposed as one of the parameters to monitor patients receiving allergen-specific immunotherapy [4]. As in the case of allergen specific IgG, however, there is no descriptive reference profile for the specific IgG4 subtype. Therefore, the purpose of the present study was to establish reference values for six common inhalant allergens in a paediatric population of two age groups (1–3 versus 7–12) testing whether there are significant differences related to gender, different allergen types (seasonal versus perennial). 2. Methods and statistical analysis Two hundred and thirteen healthy children living in Italy, in rural or urban areas were evaluated. The age and gender distributions of the study population included the following: 60 subjects were from one to three years (30 males, 30 females); 153 subjects were from seven to 12 years (77 males, 76 females). The family histories in first-degree relatives and personal histories were negative for allergic diseases (i.e. atopic dermatitis, wheezing/asthma, and rhinoconjunctivitis). All subjects were negative to both the skin prick test and an in vitro IgE screening test for allergy. The sera of all patients were drawn outside of the seasonal pollination period. The study was approved by the institutional review board of the University of Pavia and informed consent was obtained from at least one parent of each patient before blood was drawn.
411
Serum was separated from all blood samples within four hours and stored at –208 C until testing. Determination of allergen-specific IgG4 and total IgG antibodies was performed on all sera against the following six common inhalant allergens: Dermatophagoides pteronyssinus (D1), Dermatophagoides farinae (D2), Phleum pratense (G6), cat dandruff (E1), dog dandruff (E5), and Parietaria judaica (W21) in a single laboratory session (Phadia AB, Uppsala, Sweden) using an IgG RAST FEIA assay described previously [5]. Briefly, the method consists of the following steps. The test serum was added to the ImmunoCAP (a solid phase to which the allergen of interest is covalently bound) and incubated. After washing to eliminate any non-specific IgG4 or IgG, enzyme-labelled anti-IgG4 or IgG was added to form a complex. After another incubation of 150 min, any unbound labelled anti-IgG4 or IgG antibodies were eliminated by washing. The labelled allergen-antibody complex was then incubated with a developing agent and, after stopping the reaction, the fluorescence of the eluate was measured by fluorimetry (FluoroCount); and specific antibody concentrations were expressed in percentage (fluorescence sample 100/fluorescence reference serum). The higher the response, the more specific IgG or IgG4 is present in the specimen. To evaluate the test results, the response for patient samples were compared directly with a reference serum run in parallel. Since children were sampled at school, individual age values were estimated as the median of pre-school or elementary school age. Since IgG and IgG4 for all the allergens were measured using a fluorescence percentage with respect to a unique reference value and in a single laboratory session, two further measurements were calculated to minimize sampling error. 3. Statistical analysis Descriptive statistics (5th, 50th and 95th percentiles and distribution mode) have been used to characterise the sample for each allergen. The non-parametric Wilcoxon test was used to test for differences between groups, identified by gender, sex, and allergen type. The package S-Plus (MathSoft Corp) was used for all analyses. 4. Results The median, 5th and 95th percentiles of IgG and IgG4 for the six allergens are shown in Table 1. Measurement of IgG responses revealed a high variability for house dust and dog dandruff (D1, D2 and E5) with respect to a lower variability for E1, G6, W21. A significant gender difference was observed for D1 ( p = 0.04), E1 ( p = 0.02) and E5 ( p = 0.005). In the measurement of IgG4 responses, G6 appeared to have a lower variability in comparison responses seen to the other allergens. When gender data were evaluated separately, significant differences were observed for D1 ( p = 0.04) and E1 ( p = 0.002). A significant difference ( p < 0.00001) was noted between the IgG average value of the two seasonal (median 28, interquartile range 12.1–57.4) and the four perennial allergens
412
M. De Amici et al. / Revue française d’allergologie 49 (2009) 410–415
Table 1 Specific IgG and IgG4 for the six allergens in the whole sample and for males and females. Median (5, 95th percentile) Specific IgG All D1 D2 E1 E5 G6 W21 a
46.9 38.8 30.3 36.9 20.4 33.4
Specific IgG4 Male
(16.7, 106.8) (15.9, 87.7) (12.4, 60.7) (15.2, 87.1) (9.9, 42.8) (13.5, 67.6)
46.2 37.5 28.1 30.3 20.8 32.6
Female (11.8, 104.4)a (13.1, 88.2) (9.2, 61.5)a (15, 84.4)a (10.3, 41) (10.9, 67)
47.6 40.5 31.3 40.3 20.1 35.8
(22.4, 110.2)a (20.7, 58.8) (17.9, 58.8)a (17, 88.4)a (9.8, 47.4) (15, 71.7)
All 4.6 4.5 8.1 8.6 3.7 4.1
Male (2.7, (2.8, (3.2, (3.8, (2.6, (2.4,
21.1) 19.1) 18.4) 21.4) 11.6) 15.8)
4.3 4.6 6.3 8.5 3.7 4.1
(2.5, (2.8, (2.8, (3.7, (2.5, (2.3,
Female 19.4)a 19.3) 18.4)a 20.8) 12.1) 16.2)
4.7 4.5 8.8 8.7 3.6 4.2
(3.2, (2.9, (3.8, (4.1, (2.7, (2.7,
23)a 17.7) 16.6)a 27.6) 10.7) 13.8)
Significant difference between males and females.
Fig. 1. Temporal trend of specific IgG and IgG4 in males (30 subjects from one to three years and 77 from seven to 12 years) and females (30 subjects from one to three years and 76 from seven to 12 years).
M. De Amici et al. / Revue française d’allergologie 49 (2009) 410–415
(median 38, interquartile range 15.8–79.7), and this difference was also observed when males and females were compared separately. Similarly, a significant difference ( p < 0.00001) was also observed between the IgG4 average value of the two seasonal (median 4.01, interquartile range 2.71–13.51) and the four perennial allergens (median 6.55, interquartile range 3.43– 19.7), and again this difference held when considering males and females separately. Shown in Fig. 1 is a comparison of the temporal trend of specific IgG and IgG4 (medians) in males and females. Although there was no consistent pattern, some allergens increased and others decreased from pre-school to elementary school age. In females, the decrease was less evident while an increase was more evident. The Wilcoxon tests between the two age classes confirmed this observation. For example, the value 0.002d in the first cell of the table indicates that specific IgG for D1 in males decreased significantly with age ( p = 0.002). Another observation was related to IgG4 responses to dog and cat dandruff, which was clearly higher than responses to the other allergens in both genders. In Fig. 1, it is also notable that temporal trends for IgG, mainly for males, are very different
413
from IgG4 (only G6 shows a similar behaviour). In females, only E1 and G6 showed different trends. In the light of the results reported in Fig. 1, it can be argued that different reference values are necessary for the two age classes for: specific IgG D1, D2, E1, E5 and W21 in males; specific IgG E5 and G6 in females and specific IgG4 E1 and E5 in females. Percentiles of these nine allergens that significantly differ in the two age classes are shown in Fig. 2. Moreover, the difference between the distribution of the average value of perennial and seasonal allergens in the whole sample was also investigated. Perennial allergens showed higher values, the IgG distribution mode was approximately 35% (fluorescence value) for perennial, and approximately 25% for seasonal allergens, whereas the IgG4 distribution mode was approximately 5% for perennial, and around 2.5% for seasonal allergens (Fig. 3). 5. Discussion It has been previously demonstrated that allergic and healthy subjects differ not only in the presence/absence of specific IgE, but also in the preferential expression of distinct IgG
Fig. 2. Percentiles of allergens that significantly differ in the two age classes. p-values from Wilcoxon test comparing the allergens in the two age classes. Pedix specifies decreasing (d) or increasing (i) trend.
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M. De Amici et al. / Revue française d’allergologie 49 (2009) 410–415
Fig. 3. Distributions of the average values of perennial and seasonal allergens.
specificities [1-4,6]. Allergic inflammation is a systemic process involving not only the affected allergen exposed tissue but also the body as a whole, with T and B lymphocytes playing a primary role in inflammation [7]. Recent studies have provided a better understanding of the role of IgG and IgG4 during specific immunotherapy [4,8]. Studies have investigated the clinical significance of food allergen-specific IgE/IgG4 [2]. Although food allergy is known to be mediated by specific IgE antibody, the definitive role of IgG4 in food allergy is unclear. Not only allergic patients have an individual profile, but also healthy subjects have precise defined characteristics. In this context, the present work assesses reference values for the healthy paediatric population. The results of the present study show that specific IgG4 and IgG serum levels in healthy children have considerable variability to different degrees with respect to age as well as gender. Variability with age could be due to a more prolonged exposure to perennial allergens with increasing age. Indeed, there is a different pattern for pollens and perennial allergens. This hypothesis seems to be supported by our result that perennial allergens increase more than seasonal allergens with age. However, a weakness of this observation is based on the fact that two evaluated allergens are pollens, such as grasses and Parietaria. Pollen exposure may influence the levels of IgE production. We were aware of this issue and we performed the study outside the pollen season, enrolling subjects in a brief period. Therefore, this bias could be partially reduced.
In any case, this preliminary study provides evidence of a relevant variability in the IgG and IgG4 profile for common allergens in a normal population of children. The relationship of these values with those for allergic children will require future studies to establish whether the findings of our study will have clinical relevance. Consequently, the usefulness of the individual profile in clinical practice will require validation in larger randomized studies. In conclusion, the results of the present study have suggested age and gender-related differences in specific humoral responses to allergens in a normal healthy population of children further studies comparing established references values with values of allergic children will be required to establish the usefulness of this research. Acknowledgments The authors wish to thank Vania Giunta (Dipartimento di Informatica e Sistemistica, Università di Pavia) for data analysis and Laurene Kelly for correction of the English. References [1] Michils A, Vervier I, Choufani G, et al. Relationship between allergic status and specificity of IgG antibody to inhaled allergens: the grass pollen model. Clin Exp Allergy 1999;29:832–9. [2] Noh G, Ahn HS, Cho NY, et al. The clinical significance of food specific IgE/IgG4 in food specific atopic dermatitis. Pediatr Allergy Immunol 2007;18:63–70.
M. De Amici et al. / Revue française d’allergologie 49 (2009) 410–415 [3] Ling EM, Smith T, Nguyen XD, et al. Relation of CD4+CD25+ regulatory T-cell suppression of allergen-driven T-cell activation to atopic status and expression of allergic disease. Lancet 2004;363: 608–15. [4] Passalacqua G, Durham SR. Allergic rhinitis and its impact on asthma: allergen immunotherapy. J Allergy Clin Immunol 2007;119:881–91. [5] Meier P, Muller U. Evaluation of IgG RAST FEIA for the assay of venomspecific IgG antibodies during venom immunotherapy. Int Arch Allergy Immunol 1998;117. 46–51.
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[6] Duchateau J, Michils A, Michel O, et al. Mite allergy is associated with a specific profile of IgG epitopes recognized on antigen p1 of Dermatophagoides pteronyssinus. Clin Exp Allergy 1997;27:296–305. [7] Kallinich T, Beier KC, Wahn U, et al. T-cell co-stimulatory molecules: Their role in allergic immune reactions. Eur Respir J 2007;29:1246–55. [8] Rossi RE, Monasterolo G, Coco G, et al. Evaluation of serum IgG4 antibodies specific to grass pollen allergen components in the follow up of allergic patients undergoing subcutaneous and sublingual immunotherapy. Vaccine 2007;25:957–64.