- Email: [email protected]
IgE anti-respiratory syncytial virus antibodies detected in serum of pediatric patients with asthma
Human Immunology 76 (2015) 519–524
Contents lists available at ScienceDirect
www.ashi-hla.org
journal homepage: www.elsevier.com/locate/humimm
IgE anti-respiratory syncytial virus antibodies detected in serum of pediatric patients with asthma Tamar A. Smith-Norowitz a,b,⇑, Mira Mandal a, Rauno Joks b,c, Levana T. Norowitz a, Diana Weaver a, Helen G. Durkin b,d, Martin H. Bluth e, Stephan Kohlhoff a,b a
Department of Pediatrics, S.U.N.Y. Downstate Medical Center, Brooklyn, NY, 11203, United States Center for Allergy and Asthma Research, S.U.N.Y. Downstate Medical Center, Brooklyn, NY, 11203, United States Dept. of Medicine, S.U.N.Y. Downstate Medical Center, Brooklyn, NY, 11203, United States d Dept. of Pathology, S.U.N.Y. Downstate Medical Center, Brooklyn, NY, 11203, United States e Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48201, United States b c
a r t i c l e
i n f o
Article history: Received 11 March 2015 Revised 11 May 2015 Accepted 2 June 2015 Available online 11 June 2015 Keywords: Respiratory syncytial virus IgE Asthma
a b s t r a c t Respiratory syncytial virus (RSV) causes lower respiratory tract disease in infants and young children, and is a public health concern, as is the increase in pediatric asthma. Respiratory viral infections may trigger asthma exacerbations. However, it remains unknown whether RSV infection may have a specific association with asthma. Total serum IgE, and IgE- and IgG-anti-RSV Ab responses were studied in older asthmatic compared with non-asthmatic children (M/F, mean age: 14) (N = 30, N = 43, respectively). We found: (1) total serum IgE was higher in asthmatic compared with non-asthmatics (P < 0.001); (2) total serum IgE did correlate with IgE anti-RSV Abs (P < 0.001), and with IgG anti-RSV Abs (P = 0.008) in all subjects; (3) total serum IgE levels did correlate with IgE anti-RSV in asthmatics (P = 0.047), but not in non-asthmatics (P = 0.13); (4) IgE anti-RSV Abs did correlate with IgG anti-RSV Abs in all subjects (P = 0.001); (5) IgE- and IgG-anti RSV Abs were higher in asthma compared with no asthma (P = 0.003; <0.001, respectively); (6) there was a significant association between age and IgE anti-RSV in non-asthma (P = 0.008), but not in asthma (P = 0.64). Our findings indicate that IgE-anti-RSV Ab responses may play important roles in RSV infection and asthma. Ó 2015 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
1. Introduction Respiratory syncytial virus (RSV) causes severe lower respiratory tract disease (pneumonia and bronchiolitis) in infants, young children, and the elderly [1–3], and is a major public health concern. The immunopathology associated with RSV infection remains unclear, and might include immunological responses to the virus [4], including induction of virus-specific IgE and IgG4 production [5,6]; studies have reported that these Igs may contribute to development of wheezing later in life [1,7]. It has been suggested that induction of specific IgE and IgG4 responses might result from either a predisposition of the infected Abbreviations: RSV, respiratory syncytial virus; Abs, antibodies; Ig, immunoglobulin; IgG, immunoglobulin G; IgE, immunoglobulin E; ELISA, enzyme linked immunosorbent assay; TBS, tris buffered saline; TMB, 3,30 ,5,50 -tetramethyl bemzidine; HIV, human immunodeficiency virus. ⇑ Corresponding author at: Dept. of Pediatrics, Box 49, 450 Clarkson Ave., Brooklyn, NY 11203, United States. Tel.: +1 (718) 270 1295; fax: +1 (718) 270 3289. E-mail address: [email protected] (T.A. Smith-Norowitz).
infant to produce IgE Abs or from unknown effects of RSV on the host responses [8]. However, the pathogenesis of RSV-induced bronchiolitis and wheezing is not well understood, and may result from a type 1 allergic IgE mediated response, and not due to direct virus damage [9]. It should be noted that wheezing appears to be more common during rhinovirus and RSV than other virus infections in children presenting with respiratory infections, but in asthmatics wheezing occurs in 50% of viral infections [10]. The role of RSV in asthma development remains controversial [11]. Observational studies have described an association between RSV infection in infants and higher rates of bronchiolitis and recurrent wheeze in early childhood [11]. Zomer-Kooijker et al. demonstrated increased risk of wheeze and decreased lung function after RSV infection [12]. Similarly, studies of Sigurs et al. also reported that severe early RSV bronchiolitis is associated with an increased prevalence of allergic asthma persisting into early adulthood [13]. Although the literature has reported viruses as precipitants of asthma symptoms [10], there have been few clinical studies that assess trends of RSV-infection on diseases associated with
http://dx.doi.org/10.1016/j.humimm.2015.06.002 0198-8859/Ó 2015 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
520
T.A. Smith-Norowitz et al. / Human Immunology 76 (2015) 519–524
IgE/inflammatory responses (i.e. asthma) in older children, the existence of long-term persistence of IgE anti-RSV responses, or its possible role in protective immunity. In this study we investigated whether RSV, which is a known respiratory virus [1–3], can be an infectious trigger for asthma. Human RSV was chosen because it is the most common cause of severe bronchiolitis in infants [14]; it is well known that early viral lower respiratory tract infections are usually followed by recurrent wheeze, asthma and allergy in childhood [15,16] Thus, in the current study we evaluated whether RSV infection may have a specific association with asthma. 2. Materials and methods 2.1. Study population We conducted a cross-sectional study that was part of a larger investigation into allergic asthma and RSV infection in older children in Brooklyn, NY. We recruited children from among patients at the Pediatric Asthma and General Pediatric Clinics at Kings County Hospital Center (Brooklyn, NY, USA) and Lutheran Medical Center (Brooklyn, NY, USA) (See Section 3). Criteria for eligibility were: physician’s diagnosis of asthma or current clinically defined persistent asthma symptoms [17], or both, and allergic rhinoconjunctivitis. Subjects were nonsmokers, had no chest infection or antibiotic use within the past month, and were human immunodeficiency virus (HIV) negative. Asthma subjects (45%) received daily treatment with a controller medication. Exclusion criteria were: history of autoimmune disorders, recent use of systemic corticosteroids (less than 30 days), or immunotherapy, personal or family history of cigarette smoking or tobacco use, and incomplete follow-up. Control subjects were recruited from an outpatient pediatric practice (Brooklyn, NY), and had no history of doctor-diagnosed asthma, as defined by absence of asthma and/or atopy, based on clinical criteria [18]. RSV Ab titer levels were confirmed by positive anti-RSV IgG Ab levels (<8 U/mL, Negative; 8–12 U/mL, Equivocal; >12 U/mL, Positive) (ELISA). The protocol was approved by the SUNY Downstate Medical Center Institutional Review Board, and the procedures followed were in accordance with institutional guidelines involving human subjects. The enrolled participants or their guardians provided written informed consent. 2.2. Blood Peripheral blood (1–3 mL) samples were collected from asthmatic patients (N = 30) and non asthmatic controls (N = 43) (males/females; ages 1–21 years old). 2.3. Ig determination: total serum IgE Blood was collected and IgE levels were determined in serum using the UniCap Total IgE fluroenzyme immunoassay (Phamacia and Upjohn Diagnostics) performed according to the manufacturer’s recommendations (Reference range for healthy serum: 20–100 IU/mL). All tests were performed in the Clinical Diagnostic Laboratory at SUNY Downstate Medical Center (Brooklyn, NY).
reported as U/mL. (Ranges for RSV Ab IgG: Negative <8.0; Equivocal: 8–12; Positive: >12.0). 2.4.2. IgE The presence of IgE anti-RSV Abs was determined by a modification of ELISA using IgG RSV ELISA kits (IBL America). Briefly, samples were directly added (100 ll) to the microwell plates pre-coated with RSV antigen and incubated for 1 h RT. Goat polyclonal anti-human IgE (100 ll) (ICN Biomedicals, Aurora, OH), diluted 1:200 in TBS wash buffer (Tris Buffered Saline-0.05% Tween) was added to each well, and incubated for 1 h. The wells were washed 3 in wash buffer. Rabbit anti-goat peroxidase-labeled antibody (ICN Biomedicals), diluted 1:1000 in washing/diluting buffer, was then added to each well and incubated for 1 h. The wells were washed again 3 in washing/diluting buffer, and developed in 3,3 0 ,5,50 -tetramethylbemzidine (TMB) substrate solution (100 ll) (Bio-Quant, San Diego, CA) for 10 min. The reaction was stopped by adding 1 N H2SO4 (100 ll). Samples were run in duplicate. The plates were read using an automated microplate reader (Model Elx800; Bio-Tek Instruments, Winooski, VT); optical density (O.D.) measurements were read at 450 nm. For determination of RSV IgE, data are reported as O.D. values (range: positive cutoff value >0. 435 O.D. Value, positive). Final O.D. value reported was subtracted from chromagen blank O.D. value (background). 2.4.3. Statistical methods Ig levels were presented as median values ± standard deviation (SD). Spearman tests were used for nonparametric data analysis to compare antibody levels of IgG- and IgE-anti-RSV in all subjects. Kruskal–Wallis tests were used for nonparametric data analysis to compare immunoglobulin levels between asthmatics and non-asthmatics. Anti-RSV Abs were dichotomized using predefined assay thresholds (IgG: >12 U/mL) and differences of positivity were assessed in asthmatics compared with non-asthmatics using Chi-Square tests. All data and statistical analysis were performed using SAS software version 9.2 (SAS Institute, Cary, N.C.). A two-sided P-value <0.05 was considered significant. 3. Results 3.1. Subjects A total of 68 subjects were recruited into the study; 28 (41%) were asthmatic patients and 40 (59%) were non-asthmatic controls. Gender (50% male, 50% female); and age distribution were similar between asthma and non asthmatic subjects mean age (yrs): 14.0 ± 3.5, 13.4 ± 5.5, respectively; P = 0.55) Asthmatic subjects had significantly higher total serum IgE Ab levels than non-asthmatic subjects (323 IU/mL ± 217, 108 ± 119, respectively; P < 0.001). 3.2. Correlation between total serum IgE levels and IgE- or IgG- anti RSV Abs Total serum IgE levels did correlate with IgE-anti-RSV Abs (Pearson correlation, R = 0.486, P < 0.001) (Fig 1A), and with IgG-anti-RSV Abs (Pearson correlation, R = 0.341, P = 0.008) in all subjects (Fig 1B).
2.4. RSV serum Ab detection: enzyme linked immunosorbent assay 2.4.1. IgG The presence of IgG anti-RSV Abs was determined by enzyme linked immunosorbent assay (ELISA) (IBL America, Minneapolis, MN) according to manufacturer’s recommendations. Data are
3.3. Correlation between total serum IgE level and IgE-anti-RSV Abs according to asthma status There was a positive correlation found between total serum IgE levels and IgE-anti RSV Abs in asthmatic patients (Pearson
T.A. Smith-Norowitz et al. / Human Immunology 76 (2015) 519–524
521
Fig. 1. Correlation between total serum IgE levels and IgE- or IgG-anti RSV Abs. Asthmatic patients (N = 28) and non asthmatic subjects (N = 40). Top panel: (A) IgE anti RSV Abs (Pearson correlation, R = 0.486, P < 0.001); lower panel: (B) IgG anti RSV Abs (Pearson correlation, R = 0.341, P = 0.008).
correlation, R = 0.428, P = 0.047), but not in non asthmatic subjects (Pearson correlation; R = 0.25, P = 0.13) (Fig 2). 3.4. Correlation between levels of specific IgE- and IgG anti-RSV Abs There was a positive correlation between specific IgE- and IgG-anti RSV Abs in asthmatic and non asthmatic subjects (Pearson correlation, R = 0.431, P = 0.001) (Fig 3).
116 ± 30, 75 ± 46, P < 0.001, respectively) (Fig 4A and B). All subjects had positive IgG-anti-RSV titers. 3.6. Association between age and IgE-anti-RSV Abs There was a significant association between age and IgE anti RSV Abs in patients without asthma (R = 0.42, P = 0.008), but not in patients with asthma (R = 0.104, P = 0.64) (Fig 5). 4. Discussion
3.5. Differences in IgE- and IgG-anti-RSV Ab levels in asthma compared with no asthma IgE- and IgG-anti RSV Abs were significantly higher in asthma compared with no asthma (0.90 ± 0.4, 0.57 ± 0.34, P = 0.003;
This study provides new findings regarding the association between RSV infection and IgE responses in children with asthma. We found that: (1) there were correlations between total serum IgE levels and specific IgE and IgG anti-RSV Abs in all subjects;
522
T.A. Smith-Norowitz et al. / Human Immunology 76 (2015) 519–524
Fig. 2. Correlation between total serum IgE levels and IgE anti RSV Abs according to asthma status. Asthmatic patients (N = 28) (Pearson correlation, R = 0.428, P = 0.047), and non asthmatic subjects (N = 40) (Pearson correlation; R = 0.25, P = 0.13). Asthma: closed circle, solid line. No asthma: open circle, dotted line.
with viral antigen levels in secretions post infection, but did not correlate with wheezing [19]. In contrast, Bui et al., demonstrated the presence of IgE anti-RSV in serum of infants with acute RSV infection, which correlated with wheezing and bronchiolitis [5]. However, others were unable to demonstrate anti-RSV specific IgE Abs in the nasopharyngeal secretions of infants with RSV bronchiolitis [9], or a relationship between wheezing and IgE-anti-RSV [9,20]. It has been reported that infants with highest IgE-anti-RSV responses had more wheezing episodes than infants who did not have IgE anti-RSV responses [21]. However, the results of our current study are consistent with previous reports that found a possible relationship between RSV-specific IgE response at the time of infection and subsequent wheezing [22], and that RSV infection is frequently observed in patients with acute bronchiolitis [23]. But, it should be mentioned, due to our cross-sectional study design, we were unable to determine whether the above differences in subjects with asthma were due to recurrent or prolonged infections. A possible explanation might be that RSV infection might stimulate induction of genes associated with recurrent wheezing [24] or it could be that individuals with abnormal lung function are more susceptible to RSV infection. Our data show the association between RSV-specific-IgE and IgG responses in older children, which to our knowledge, has not been previously studied. Given the peak incidence of RSV infection is in infancy, our results indicate that there is an increased number
(2) there was a correlation between total serum IgE and IgE anti-RSV Abs in asthmatic patients but not in non asthmatic patients (3) there was a correlation between specific IgE and specific IgG; (4) asthmatic subjects had higher specific IgE- and IgG anti RSV Abs compared with non asthmatic subjects, and (5) there was an association between age and IgE anti-RSV in patients without asthma. Thus, our findings indicate relevant involvement of IgG- and IgE-anti-RSV Abs in asthma. Our main finding was that asthmatic subjects had higher levels of IgE- and IgG-anti-RSV Abs compared with non-asthmatic subjects. These results are in agreement with findings from prior literature that reported that IgE may be involved in the pathogenesis of RSV infection and that IgE Abs bound to RSV-infected cells in nasal secretions of acute RSV-infected infants, but not uninfected infants [19]. However, putative IgE anti-RSV concentrations correlated
Fig. 3. Correlation between IgE- and IgG-anti-RSV Abs. Specific IgE- and IgG-antiRSV Ab levels were similar in asthmatic patients (N = 28) and non asthmatic subjects (N = 40) (Pearson correlation, R = 0.431, P = 0.001).
Fig. 4. Differences in IgE- and IgG-anti-RSV Ab levels in asthma compared with no asthma. Top panel: (A) IgE-anti-RSV Abs were significantly higher in asthmatic patients compared with non-asthmatic subjects (P = 0.003). Lower panel: (B) IgG anti-RSV Abs were significantly higher in asthmatic patients compared with nonasthmatic subjects (P < 0.001).
T.A. Smith-Norowitz et al. / Human Immunology 76 (2015) 519–524
Fig. 5. Association between age and IgE Abs. Significant association between age and IgE-anti RSV Abs in non-asthmatic subjects (R = 0.42, P = 0.008), but not in asthmatic patients (R = 0.104, P = 0.64).
of RSV infections in older children with asthma, but not in children without asthma, who might be immune to infection. If the RSV antigen is responsible for activating the IgE response in patients with asthma, this response presumably gets reactivated, and as a result, more IL-4 or IL-13 is produced. IL-4 is responsible for continuation of Th2 responses [25], which plays a role in mediating allergic/IgE responses [25]. Another possibility may include a genetic component which might prevent the response(s) from turning off, perhaps by preventing the production of suppressor cells. Thus, exposure to RSV boosts a response which is helped by excess IL-4. On the basis of these results, we suggest that the inflammatory component of diseases of altered IgE responses (e.g. asthma) may be strongly driven by specific IgE-anti-viral responses. The putative role of inflammation has previously been described in several diseases such as atopic dermatitis, eczema, and asthma [26], but our results bring a viral underpinning to these observations. It could be that impaired immune responses, which might include dysfunction of T regulatory cells, decrease in the levels of regulatory cytokines or change in number of CD4+ T cells (producing IL-4 or IL-13), may be possible explanations for viral pathogenesis, asthma inflammation or health. Despite increasing scientific research on the relationship between RSV infection and IgE mediated responses, the mechanisms and functions remain elusive. Thus, the functionality of these IgE responses needs to be investigated. Another finding in the current study revealed that total serum IgE levels did correlate with IgE-anti-RSV Ab levels in patients with asthma but not in subjects without asthma. Thus, the levels of antigen specific IgE in the total IgE pool may be responsible for responses compared with total IgE levels alone [27], which may be differentially regulated in asthma. It could be that the hyper-inflammatory status of the asthmatic patient facilitates the propagation of RSV specific IgE (and IgG?) either in conjunction with total IgE levels or in a manner independent of IgE pathobiology. Differential IgE and IgG responses in relation to T cell responses have been reported in other IgE mediated diseases [28] as well as in immunodeficiency states [29], implying that antigen specific IgE may or may not be coupled to total IgE levels and the relationship may differ depending on disease status. It should also be mentioned that RSV infection can induce production of
523
RSV-specific IgG4 Abs in children [5,6]; however, IgG4 anti-RSV antibodies were not studied in the current investigation. It could be speculated that the IgE anti-RSV Abs are protective in asthma through unknown mechanisms. Previous studies in our laboratory demonstrated that IgE may play an important role in immunity to specific viruses including Parvovirus B19 [30], Varicella Zoster Virus [31,32], Influenza Virus [33] and HIV-1 [34,35] In addition, earlier studies in our laboratory found that serum from certain HIV-1 infected pediatric long-term survivors contain agents that inhibit HIV-1 production in vitro, including anti-HIV-1 IgE [34]; it has been suggested that cytotoxicity plays a role in anti-HIV-1 IgE-mediated inhibition of virus production [35] Thus, these specific IgE responses may play an important role in viral memory responses, and provide protection against viral infection. Another notable finding in the current study was that there was a significant association between IgE-anti-RSV and age in non-asthmatic subjects, but not in asthmatic subjects. The association between age and IgE-anti- RSV Abs has not been widely studied. However, the current finding has significant clinical implications. It could be that in asthma the B cells are already primed due to the inflammatory state and as such, the titer level of IgE-anti-RSV is also up-regulated, as has been shown in selective immunoglobulin responses in other antigen stimulation settings [36] In patients with asthma the lack of correlation of age might be due to reaching a threshold of responses, where once attained, there is no clear feedback loop to regulate this response with time (e.g. age), or alternatively, it could be that it is contingent on the age of onset in addition to the time of antigen exposure and maternal transfer of Ab (blood or breast feeding), which may govern these collective responses [37]. However, subjects without asthma may possess a more linear response to antigen exposure, due to lack of hyper inflammations and potential dysregulation which exists in the asthmatic state [38,39] As such, the non asthmatic T/B cell profile or TH1/Th2 response may not have yet reached the threshold limits (since the virus specific Ab responses were actually lower than that of asthmatic patients). Or it could be in non asthma, there may be more responsiveness to potentiating virus specific antibody signals (e.g. cytokines) than that of the asthmatic patient. Such differences in cytokine responses in asthma compared with no asthma compared with atopic patients have been reported [39,40]. Others have reported excess Th2 –type cytokine immune responses, with deficient Th1 type responses, in RSV-infected infants [41], which may result in a more severe course of infection; Th1 type responses are required for efficient antiviral responses [41]. This study has limitations that should be considered in interpreting these findings, including: (1) modest sample size, and (2) recurrent wheezing in children is not always asthma, but might occur after viral infection. Despite these limitations, our study has carefully structured experimental research design and statistically significant findings. This study provides evidence of an association between RSV Abs in asthmatic children and IgE responses, and underscores the importance of IgE and viruses affecting the health of children with asthma. Future research efforts are necessary to develop new and effective therapeutics to prevent RSV disease, as well as testing new immunological biomarkers. Disclosure The authors declare no competing financial interest to disclose. Conflict of interest The authors declare no conflict of interest to disclose.
524
T.A. Smith-Norowitz et al. / Human Immunology 76 (2015) 519–524
Acknowledgments We thank Kevin B. Norowitz, M.D., (SUNY Downstate Medical Center, Department of Pediatrics) for helpful comments and thoughtful discussion. This study was funded by a NY State Divisional Grant/Pilot Project Grant. References [1] Everard ML, Milner AD. The respiratory syncytial virus and its role in acute bronchiolitis. Eur. J. Pediatr. 1992;151:638–51. [2] Hortal M, Russi JC, Arbiza JR, Canepa E, Chiparelli H, Illaramendy A. Identification of viruses in a study acute respiratory tract infection in children in Uruguay. Rev. Infect. Dis. 1990;12(Suppl. 8):S995–7. [3] Falsey AR, Hennessey PA, Formica MA, Cox C, Walsh EE. Respiratory syncytial virus infection in elderly and high risk adults. N. Engl. J. Med. 2005;352: 1749–59. [4] McIntosh K, Fishaut JM. Immunopathologic mechanisms in lower respiratory tract disease of infants due to respiratory syncytial virus. Prog. Med. Virol. 1980;26:94–118. [5] Bui RH, Molinaro GA, Kettering JD, Heiner DC, Imagawa DT, St Geme Jr JW. Virus-specific IgE and IgG4 antibodies in serum of children infected with respiratory syncytial virus. J. Pediatr. 1987;110:87–90. [6] Welliver RC, Sun M, Hildreth SW, Arumugham R, Ogra PL. Respiratory syncytial virus-specific antibody responses in immunoglobulin A and E isotypes to the F and G proteins and to intact virus after natural infection. J. Clin. Microbiol. 1989;27:295–9. [7] Bardin PG, Johnston SL, Pattermore PK. Viruses as precipitants of asthma symptoms. II. Physiology and mechanisms. Clin. Exp. Allergy 1992;22:809–22. [8] Rabatic S, Gagro A, Lokar-Kolbas R, Krsulovic-Hresic V, Vrtar Z, Popow-Kraupp T, et al. Increase in CD23+ B cells in infants with bronchiolitis is accompanied by appearance of IgE and IgG4 antibodies specific for respiratory syncytial virus. J. Infect. Dis. 1997;175:32–7. [9] Toms GL, Quinn R, Robinson JW. Undetectable IgE responses after respiratory syncytial virus infection. Arch. Dis. Child. 1996;74:126–30. [10] Pattenmore PK, Johnston SL, Bardin PG. Viruses as precipitants of asthma symptoms, I epidemiology. Clin. Exp. Allergy 1992;22:325–36. [11] Blanken MO, Rovers MM, Molenaar JM, Winkler-Seinstra PL, Meijer A, Kimpen JL, et al. Respiratory syncytial virus and recurrent wheeze in healthy preterm infants. N. Engl. J. Med. 2013;368:1791–9. [12] Zomer-Kooijker K, van der Ent CK, Ermers MJ, Uiterwaal CS, Rovers MM, Bont LJ, RSV Corticosteroid Study Group. Increased risk of wheeze and decreased lung function after respiratory syncytial virus infection. PLoS One 2014;9(1):e87162. http://dx.doi.org/10.1371/journal.pone.0087162. [13] Sigurs N, Aljassim F, Kjellman B, Robinson PD, Sigurbergsson F, Bjarnason R, Gustafsson PM. Asthma and allergy patterns over 18 years after severe RSV bronchiolitis in the first year of life. Thorax 2010;65:1045–52. [14] Smyth RL, Openshaw PJ. Bronchiolitis. Lancet 2006;368:312–22. [15] Gelfand EW. Pediatric asthma: a different disease. Proc. Am. Thorac. Soc. 2009;6:278–82. [16] Walton RP, Johnston SL. Role of respiratory viral infections in the development of atopic conditions. Curr. Opin. Allergy Clin. Immunol. 2008;8:150–3. [17] Cowen MK, Wakefield DB, Cloutier MM. Classifying asthma severity: objective versus subjective measures. J. Asthma 2007;44:711–5. [18] EPR-3, Expert panel report 3: guidelines for the diagnosis and management of asthma (EPR-3 2007), U.S. Department of Health and Human Services; National Institutes of Health; National Heart, Lung, and Blood Institute; National Asthma Education and Prevention Program, 2007; 40–3. http://www. nhlbi.nih.gov/guidelines/asthma/asthgdln.pdf. [19] Russi JS, Delfraro A, Borthagaray MD, Velazquez B, Barreno G, Hortal M. Evaluation of immunoglobulin E-specific antibodies and viral antigens in nasopharyngeal secretions of children with respiratory syncytial virus infection. J. Clin. Microbiol. 1993;31:819–23. [20] De Alcaron A, Walsh EE, Carper HT, La Russa JB, Evans BA, Rakes GP, et al. Detection of IgA and IgG but not IgE antibody to respiratory syncytial virus in nasal washes and sera from infants with wheezing. J. Pediatr. 2001;138:311–7.
[21] Welliver RC, Duffy L. The relationship of RSV-specific immunoglobulin E antibody responses in infancy, recurrent wheezing, and pulmonary function at age 7–8 years. Pediatr. Pulmonol. 1993;15:19–27. [22] Welliver RC, Sun M, Rinaldo D, Ogra PL. Predictive value of respiratory syncytial virus-specific IgE responses for recurrent wheezing following bronchiolitis. J. Pediatr. 1986;109:776–80. [23] Kwon J-M, Shim JW, Kim DS, Jung HL, Park MS, Shim JY. Prevalence of respiratory viral infection in children hospitalized for acute lower respiratory tract diseases, and association of rhinovirus and influenza virus with asthma exacerbations. Korean J. Pediatr. 2014;57:29–34. [24] Kumar RK, Hitchins MP, Foster PS. Epigenetic changes in childhood asthma. Dis. Model. Mech. 2009;2:549–53. [25] Gilmour J, Lavender P. Control of IL-4 expression in T helper 1 and 2 cells. Immunology 2008;124:437–44. [26] Weidinger S, Willis-Owen SA, Kamatani Y, Baurecht H, Morar N, Liang L, et al. A genome-wide association study of atopic dermatitis identifies loci with overlapping effects on asthma and psoriasis. Hum. Mol. Genet. 2013;22(23): 4841–56. [27] Viterbo D, Callender GE, DiMaio T, Mueller CM, Smith-Norowitz T, Zenilman ME, et al. Administration of anti-Reg I and anti PAP II antibodies worsens pancreatitis. JOP 2009;10:15–23. [28] Alonso MG, Caballero ML, Umpierrez A, Lluch-Bernal M, Knaute T, RodríguezPérez R. Relationships between T-cell and IgE/IgG4 epitopes of the Anisakis simplex major allergen Ani s 1. Clin. Exp. Allergy 2014. http://dx.doi.org/ 10.1111/cea.12474 [Epub ahead of print]; PMID: 25495594. [29] Quezada A, Norambuena X, Inostroza J, Rodríguez J. Specific antibody deficiency with normal immunoglobulin concentration in children with recurrent respiratory infections. Allergol. Immunopathol. (Madr.) 2014. http://dx.doi.org/10.1016/j.aller.2014.07.009. pii: S0301-0546(14)00162-1. [Epub ahead of print] PMID: 25498324. [30] Smith-Norowitz TA, Drew H, Norowitz HM, Bluth EF, Durkin HG, Bluth MH. Detection of IgE anti parvovirus antibodies in human breast milk. Ann. Clin. Lab. Sci. 2008;38:168–73. [31] Smith-Norowitz TA, Josekutty J, Lev-Tov H, Norowitz KB, Kohlhoff SA, Silverberg JI, et al. Long term persistence of IgE anti-Varicella Zoster Virus in pediatric and adult serum post chicken pox infection and after vaccination with Varicella Virus vaccine. Int. J. Biomed. Sci. 2009:100–5. [32] Smith-Norowitz TA, Josekutty J, Lev-Tov H, Kohlhoff S, Norowitz KB, Silverberg JI, et al. IgE anti-Varicella Zoster virus and other immune responses before, during, and after shingles. Ann. Clin. Lab. Sci. 2009;39:43–50. [33] Smith-Norowitz TA, Wong D, Kusonruksa M, Norowitz KB, Joks R, Durkin HG, et al. Long term persistence of IgE anti-influenza virus antibodies in pediatric and adult serum post vaccination with influenza virus vaccine. Int. J. Med. Sci. 2011;8:239–44. [34] Secord EA, Kleiner GI, Auci DL, Smith-Norowitz T, Chice S, Finkielstein A, et al. IgE against HIV proteins in clinically healthy children with HIV disease. J. Allergy Clin. Immunol. 1996;98:979–84. [35] Pellegrino MG, Bluth MH, Smith-Norowitz TA, Fikrig S, Volsky DJ, Moallem H, et al. HIV type 1-specific IgE in serum of long-term surviving children inhibits HIV type 1 production in vitro. AIDS Res. Hum. Retroviruses 2002; 18:363–72. [36] Fu SI, Pierre J, Smith-Norowitz TA, Hagler M, Bowner W, Pincus MR, et al. Immunuoglobulin E antibodies from pancreatic cancer patients mediate antibody-dependent cell-mediated cytotoxicity against pancreatic cancer cells. Clin. Exp. Immunol. 2008;153:401–9. [37] Dias AS, Gerber PF, Araujo AS, Auler PA, Gallinari GC, Lobato ZI. Lack of antibody protection against Porcine circovirus 2 and Porcine parvovirus in naturally infected dams and their offspring. Res. Vet. Sci. 2013;94:341–5. [38] Djukanovic R. Asthma: a disease of inflammation and repair. J. Allergy Clin. Immunol. 2000;105:S522–6. [39] Pala P, Message SD, Johnston SL, Openshaw PJM. Increased aeroallergenspecific interleukin-4-producing T cells in asthmatic adults. Clin. Exp. Allergy 2002;32:1739–44. [40] Tang C, Rolland IM, Ward C, Bish R, Thien F, Walters EH. Seasonal comparison of cytokine profiles in astopic asthmatics and atopic non asthmatics. Am. J. Respir. Crit. Care Med. 1996;154:1615–22. [41] Legg JP, Hussain IR, Warner JA, Johnston SL, Warner JO. Type 1 and type 2 cytokine imbalance in acute respiratory syncytial virus bronchiolitis. Am. J. Respir. Crit. Care Med. 2003;168:633–9.
Contents lists available at ScienceDirect
www.ashi-hla.org
journal homepage: www.elsevier.com/locate/humimm
IgE anti-respiratory syncytial virus antibodies detected in serum of pediatric patients with asthma Tamar A. Smith-Norowitz a,b,⇑, Mira Mandal a, Rauno Joks b,c, Levana T. Norowitz a, Diana Weaver a, Helen G. Durkin b,d, Martin H. Bluth e, Stephan Kohlhoff a,b a
Department of Pediatrics, S.U.N.Y. Downstate Medical Center, Brooklyn, NY, 11203, United States Center for Allergy and Asthma Research, S.U.N.Y. Downstate Medical Center, Brooklyn, NY, 11203, United States Dept. of Medicine, S.U.N.Y. Downstate Medical Center, Brooklyn, NY, 11203, United States d Dept. of Pathology, S.U.N.Y. Downstate Medical Center, Brooklyn, NY, 11203, United States e Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48201, United States b c
a r t i c l e
i n f o
Article history: Received 11 March 2015 Revised 11 May 2015 Accepted 2 June 2015 Available online 11 June 2015 Keywords: Respiratory syncytial virus IgE Asthma
a b s t r a c t Respiratory syncytial virus (RSV) causes lower respiratory tract disease in infants and young children, and is a public health concern, as is the increase in pediatric asthma. Respiratory viral infections may trigger asthma exacerbations. However, it remains unknown whether RSV infection may have a specific association with asthma. Total serum IgE, and IgE- and IgG-anti-RSV Ab responses were studied in older asthmatic compared with non-asthmatic children (M/F, mean age: 14) (N = 30, N = 43, respectively). We found: (1) total serum IgE was higher in asthmatic compared with non-asthmatics (P < 0.001); (2) total serum IgE did correlate with IgE anti-RSV Abs (P < 0.001), and with IgG anti-RSV Abs (P = 0.008) in all subjects; (3) total serum IgE levels did correlate with IgE anti-RSV in asthmatics (P = 0.047), but not in non-asthmatics (P = 0.13); (4) IgE anti-RSV Abs did correlate with IgG anti-RSV Abs in all subjects (P = 0.001); (5) IgE- and IgG-anti RSV Abs were higher in asthma compared with no asthma (P = 0.003; <0.001, respectively); (6) there was a significant association between age and IgE anti-RSV in non-asthma (P = 0.008), but not in asthma (P = 0.64). Our findings indicate that IgE-anti-RSV Ab responses may play important roles in RSV infection and asthma. Ó 2015 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
1. Introduction Respiratory syncytial virus (RSV) causes severe lower respiratory tract disease (pneumonia and bronchiolitis) in infants, young children, and the elderly [1–3], and is a major public health concern. The immunopathology associated with RSV infection remains unclear, and might include immunological responses to the virus [4], including induction of virus-specific IgE and IgG4 production [5,6]; studies have reported that these Igs may contribute to development of wheezing later in life [1,7]. It has been suggested that induction of specific IgE and IgG4 responses might result from either a predisposition of the infected Abbreviations: RSV, respiratory syncytial virus; Abs, antibodies; Ig, immunoglobulin; IgG, immunoglobulin G; IgE, immunoglobulin E; ELISA, enzyme linked immunosorbent assay; TBS, tris buffered saline; TMB, 3,30 ,5,50 -tetramethyl bemzidine; HIV, human immunodeficiency virus. ⇑ Corresponding author at: Dept. of Pediatrics, Box 49, 450 Clarkson Ave., Brooklyn, NY 11203, United States. Tel.: +1 (718) 270 1295; fax: +1 (718) 270 3289. E-mail address: [email protected] (T.A. Smith-Norowitz).
infant to produce IgE Abs or from unknown effects of RSV on the host responses [8]. However, the pathogenesis of RSV-induced bronchiolitis and wheezing is not well understood, and may result from a type 1 allergic IgE mediated response, and not due to direct virus damage [9]. It should be noted that wheezing appears to be more common during rhinovirus and RSV than other virus infections in children presenting with respiratory infections, but in asthmatics wheezing occurs in 50% of viral infections [10]. The role of RSV in asthma development remains controversial [11]. Observational studies have described an association between RSV infection in infants and higher rates of bronchiolitis and recurrent wheeze in early childhood [11]. Zomer-Kooijker et al. demonstrated increased risk of wheeze and decreased lung function after RSV infection [12]. Similarly, studies of Sigurs et al. also reported that severe early RSV bronchiolitis is associated with an increased prevalence of allergic asthma persisting into early adulthood [13]. Although the literature has reported viruses as precipitants of asthma symptoms [10], there have been few clinical studies that assess trends of RSV-infection on diseases associated with
http://dx.doi.org/10.1016/j.humimm.2015.06.002 0198-8859/Ó 2015 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
520
T.A. Smith-Norowitz et al. / Human Immunology 76 (2015) 519–524
IgE/inflammatory responses (i.e. asthma) in older children, the existence of long-term persistence of IgE anti-RSV responses, or its possible role in protective immunity. In this study we investigated whether RSV, which is a known respiratory virus [1–3], can be an infectious trigger for asthma. Human RSV was chosen because it is the most common cause of severe bronchiolitis in infants [14]; it is well known that early viral lower respiratory tract infections are usually followed by recurrent wheeze, asthma and allergy in childhood [15,16] Thus, in the current study we evaluated whether RSV infection may have a specific association with asthma. 2. Materials and methods 2.1. Study population We conducted a cross-sectional study that was part of a larger investigation into allergic asthma and RSV infection in older children in Brooklyn, NY. We recruited children from among patients at the Pediatric Asthma and General Pediatric Clinics at Kings County Hospital Center (Brooklyn, NY, USA) and Lutheran Medical Center (Brooklyn, NY, USA) (See Section 3). Criteria for eligibility were: physician’s diagnosis of asthma or current clinically defined persistent asthma symptoms [17], or both, and allergic rhinoconjunctivitis. Subjects were nonsmokers, had no chest infection or antibiotic use within the past month, and were human immunodeficiency virus (HIV) negative. Asthma subjects (45%) received daily treatment with a controller medication. Exclusion criteria were: history of autoimmune disorders, recent use of systemic corticosteroids (less than 30 days), or immunotherapy, personal or family history of cigarette smoking or tobacco use, and incomplete follow-up. Control subjects were recruited from an outpatient pediatric practice (Brooklyn, NY), and had no history of doctor-diagnosed asthma, as defined by absence of asthma and/or atopy, based on clinical criteria [18]. RSV Ab titer levels were confirmed by positive anti-RSV IgG Ab levels (<8 U/mL, Negative; 8–12 U/mL, Equivocal; >12 U/mL, Positive) (ELISA). The protocol was approved by the SUNY Downstate Medical Center Institutional Review Board, and the procedures followed were in accordance with institutional guidelines involving human subjects. The enrolled participants or their guardians provided written informed consent. 2.2. Blood Peripheral blood (1–3 mL) samples were collected from asthmatic patients (N = 30) and non asthmatic controls (N = 43) (males/females; ages 1–21 years old). 2.3. Ig determination: total serum IgE Blood was collected and IgE levels were determined in serum using the UniCap Total IgE fluroenzyme immunoassay (Phamacia and Upjohn Diagnostics) performed according to the manufacturer’s recommendations (Reference range for healthy serum: 20–100 IU/mL). All tests were performed in the Clinical Diagnostic Laboratory at SUNY Downstate Medical Center (Brooklyn, NY).
reported as U/mL. (Ranges for RSV Ab IgG: Negative <8.0; Equivocal: 8–12; Positive: >12.0). 2.4.2. IgE The presence of IgE anti-RSV Abs was determined by a modification of ELISA using IgG RSV ELISA kits (IBL America). Briefly, samples were directly added (100 ll) to the microwell plates pre-coated with RSV antigen and incubated for 1 h RT. Goat polyclonal anti-human IgE (100 ll) (ICN Biomedicals, Aurora, OH), diluted 1:200 in TBS wash buffer (Tris Buffered Saline-0.05% Tween) was added to each well, and incubated for 1 h. The wells were washed 3 in wash buffer. Rabbit anti-goat peroxidase-labeled antibody (ICN Biomedicals), diluted 1:1000 in washing/diluting buffer, was then added to each well and incubated for 1 h. The wells were washed again 3 in washing/diluting buffer, and developed in 3,3 0 ,5,50 -tetramethylbemzidine (TMB) substrate solution (100 ll) (Bio-Quant, San Diego, CA) for 10 min. The reaction was stopped by adding 1 N H2SO4 (100 ll). Samples were run in duplicate. The plates were read using an automated microplate reader (Model Elx800; Bio-Tek Instruments, Winooski, VT); optical density (O.D.) measurements were read at 450 nm. For determination of RSV IgE, data are reported as O.D. values (range: positive cutoff value >0. 435 O.D. Value, positive). Final O.D. value reported was subtracted from chromagen blank O.D. value (background). 2.4.3. Statistical methods Ig levels were presented as median values ± standard deviation (SD). Spearman tests were used for nonparametric data analysis to compare antibody levels of IgG- and IgE-anti-RSV in all subjects. Kruskal–Wallis tests were used for nonparametric data analysis to compare immunoglobulin levels between asthmatics and non-asthmatics. Anti-RSV Abs were dichotomized using predefined assay thresholds (IgG: >12 U/mL) and differences of positivity were assessed in asthmatics compared with non-asthmatics using Chi-Square tests. All data and statistical analysis were performed using SAS software version 9.2 (SAS Institute, Cary, N.C.). A two-sided P-value <0.05 was considered significant. 3. Results 3.1. Subjects A total of 68 subjects were recruited into the study; 28 (41%) were asthmatic patients and 40 (59%) were non-asthmatic controls. Gender (50% male, 50% female); and age distribution were similar between asthma and non asthmatic subjects mean age (yrs): 14.0 ± 3.5, 13.4 ± 5.5, respectively; P = 0.55) Asthmatic subjects had significantly higher total serum IgE Ab levels than non-asthmatic subjects (323 IU/mL ± 217, 108 ± 119, respectively; P < 0.001). 3.2. Correlation between total serum IgE levels and IgE- or IgG- anti RSV Abs Total serum IgE levels did correlate with IgE-anti-RSV Abs (Pearson correlation, R = 0.486, P < 0.001) (Fig 1A), and with IgG-anti-RSV Abs (Pearson correlation, R = 0.341, P = 0.008) in all subjects (Fig 1B).
2.4. RSV serum Ab detection: enzyme linked immunosorbent assay 2.4.1. IgG The presence of IgG anti-RSV Abs was determined by enzyme linked immunosorbent assay (ELISA) (IBL America, Minneapolis, MN) according to manufacturer’s recommendations. Data are
3.3. Correlation between total serum IgE level and IgE-anti-RSV Abs according to asthma status There was a positive correlation found between total serum IgE levels and IgE-anti RSV Abs in asthmatic patients (Pearson
T.A. Smith-Norowitz et al. / Human Immunology 76 (2015) 519–524
521
Fig. 1. Correlation between total serum IgE levels and IgE- or IgG-anti RSV Abs. Asthmatic patients (N = 28) and non asthmatic subjects (N = 40). Top panel: (A) IgE anti RSV Abs (Pearson correlation, R = 0.486, P < 0.001); lower panel: (B) IgG anti RSV Abs (Pearson correlation, R = 0.341, P = 0.008).
correlation, R = 0.428, P = 0.047), but not in non asthmatic subjects (Pearson correlation; R = 0.25, P = 0.13) (Fig 2). 3.4. Correlation between levels of specific IgE- and IgG anti-RSV Abs There was a positive correlation between specific IgE- and IgG-anti RSV Abs in asthmatic and non asthmatic subjects (Pearson correlation, R = 0.431, P = 0.001) (Fig 3).
116 ± 30, 75 ± 46, P < 0.001, respectively) (Fig 4A and B). All subjects had positive IgG-anti-RSV titers. 3.6. Association between age and IgE-anti-RSV Abs There was a significant association between age and IgE anti RSV Abs in patients without asthma (R = 0.42, P = 0.008), but not in patients with asthma (R = 0.104, P = 0.64) (Fig 5). 4. Discussion
3.5. Differences in IgE- and IgG-anti-RSV Ab levels in asthma compared with no asthma IgE- and IgG-anti RSV Abs were significantly higher in asthma compared with no asthma (0.90 ± 0.4, 0.57 ± 0.34, P = 0.003;
This study provides new findings regarding the association between RSV infection and IgE responses in children with asthma. We found that: (1) there were correlations between total serum IgE levels and specific IgE and IgG anti-RSV Abs in all subjects;
522
T.A. Smith-Norowitz et al. / Human Immunology 76 (2015) 519–524
Fig. 2. Correlation between total serum IgE levels and IgE anti RSV Abs according to asthma status. Asthmatic patients (N = 28) (Pearson correlation, R = 0.428, P = 0.047), and non asthmatic subjects (N = 40) (Pearson correlation; R = 0.25, P = 0.13). Asthma: closed circle, solid line. No asthma: open circle, dotted line.
with viral antigen levels in secretions post infection, but did not correlate with wheezing [19]. In contrast, Bui et al., demonstrated the presence of IgE anti-RSV in serum of infants with acute RSV infection, which correlated with wheezing and bronchiolitis [5]. However, others were unable to demonstrate anti-RSV specific IgE Abs in the nasopharyngeal secretions of infants with RSV bronchiolitis [9], or a relationship between wheezing and IgE-anti-RSV [9,20]. It has been reported that infants with highest IgE-anti-RSV responses had more wheezing episodes than infants who did not have IgE anti-RSV responses [21]. However, the results of our current study are consistent with previous reports that found a possible relationship between RSV-specific IgE response at the time of infection and subsequent wheezing [22], and that RSV infection is frequently observed in patients with acute bronchiolitis [23]. But, it should be mentioned, due to our cross-sectional study design, we were unable to determine whether the above differences in subjects with asthma were due to recurrent or prolonged infections. A possible explanation might be that RSV infection might stimulate induction of genes associated with recurrent wheezing [24] or it could be that individuals with abnormal lung function are more susceptible to RSV infection. Our data show the association between RSV-specific-IgE and IgG responses in older children, which to our knowledge, has not been previously studied. Given the peak incidence of RSV infection is in infancy, our results indicate that there is an increased number
(2) there was a correlation between total serum IgE and IgE anti-RSV Abs in asthmatic patients but not in non asthmatic patients (3) there was a correlation between specific IgE and specific IgG; (4) asthmatic subjects had higher specific IgE- and IgG anti RSV Abs compared with non asthmatic subjects, and (5) there was an association between age and IgE anti-RSV in patients without asthma. Thus, our findings indicate relevant involvement of IgG- and IgE-anti-RSV Abs in asthma. Our main finding was that asthmatic subjects had higher levels of IgE- and IgG-anti-RSV Abs compared with non-asthmatic subjects. These results are in agreement with findings from prior literature that reported that IgE may be involved in the pathogenesis of RSV infection and that IgE Abs bound to RSV-infected cells in nasal secretions of acute RSV-infected infants, but not uninfected infants [19]. However, putative IgE anti-RSV concentrations correlated
Fig. 3. Correlation between IgE- and IgG-anti-RSV Abs. Specific IgE- and IgG-antiRSV Ab levels were similar in asthmatic patients (N = 28) and non asthmatic subjects (N = 40) (Pearson correlation, R = 0.431, P = 0.001).
Fig. 4. Differences in IgE- and IgG-anti-RSV Ab levels in asthma compared with no asthma. Top panel: (A) IgE-anti-RSV Abs were significantly higher in asthmatic patients compared with non-asthmatic subjects (P = 0.003). Lower panel: (B) IgG anti-RSV Abs were significantly higher in asthmatic patients compared with nonasthmatic subjects (P < 0.001).
T.A. Smith-Norowitz et al. / Human Immunology 76 (2015) 519–524
Fig. 5. Association between age and IgE Abs. Significant association between age and IgE-anti RSV Abs in non-asthmatic subjects (R = 0.42, P = 0.008), but not in asthmatic patients (R = 0.104, P = 0.64).
of RSV infections in older children with asthma, but not in children without asthma, who might be immune to infection. If the RSV antigen is responsible for activating the IgE response in patients with asthma, this response presumably gets reactivated, and as a result, more IL-4 or IL-13 is produced. IL-4 is responsible for continuation of Th2 responses [25], which plays a role in mediating allergic/IgE responses [25]. Another possibility may include a genetic component which might prevent the response(s) from turning off, perhaps by preventing the production of suppressor cells. Thus, exposure to RSV boosts a response which is helped by excess IL-4. On the basis of these results, we suggest that the inflammatory component of diseases of altered IgE responses (e.g. asthma) may be strongly driven by specific IgE-anti-viral responses. The putative role of inflammation has previously been described in several diseases such as atopic dermatitis, eczema, and asthma [26], but our results bring a viral underpinning to these observations. It could be that impaired immune responses, which might include dysfunction of T regulatory cells, decrease in the levels of regulatory cytokines or change in number of CD4+ T cells (producing IL-4 or IL-13), may be possible explanations for viral pathogenesis, asthma inflammation or health. Despite increasing scientific research on the relationship between RSV infection and IgE mediated responses, the mechanisms and functions remain elusive. Thus, the functionality of these IgE responses needs to be investigated. Another finding in the current study revealed that total serum IgE levels did correlate with IgE-anti-RSV Ab levels in patients with asthma but not in subjects without asthma. Thus, the levels of antigen specific IgE in the total IgE pool may be responsible for responses compared with total IgE levels alone [27], which may be differentially regulated in asthma. It could be that the hyper-inflammatory status of the asthmatic patient facilitates the propagation of RSV specific IgE (and IgG?) either in conjunction with total IgE levels or in a manner independent of IgE pathobiology. Differential IgE and IgG responses in relation to T cell responses have been reported in other IgE mediated diseases [28] as well as in immunodeficiency states [29], implying that antigen specific IgE may or may not be coupled to total IgE levels and the relationship may differ depending on disease status. It should also be mentioned that RSV infection can induce production of
523
RSV-specific IgG4 Abs in children [5,6]; however, IgG4 anti-RSV antibodies were not studied in the current investigation. It could be speculated that the IgE anti-RSV Abs are protective in asthma through unknown mechanisms. Previous studies in our laboratory demonstrated that IgE may play an important role in immunity to specific viruses including Parvovirus B19 [30], Varicella Zoster Virus [31,32], Influenza Virus [33] and HIV-1 [34,35] In addition, earlier studies in our laboratory found that serum from certain HIV-1 infected pediatric long-term survivors contain agents that inhibit HIV-1 production in vitro, including anti-HIV-1 IgE [34]; it has been suggested that cytotoxicity plays a role in anti-HIV-1 IgE-mediated inhibition of virus production [35] Thus, these specific IgE responses may play an important role in viral memory responses, and provide protection against viral infection. Another notable finding in the current study was that there was a significant association between IgE-anti-RSV and age in non-asthmatic subjects, but not in asthmatic subjects. The association between age and IgE-anti- RSV Abs has not been widely studied. However, the current finding has significant clinical implications. It could be that in asthma the B cells are already primed due to the inflammatory state and as such, the titer level of IgE-anti-RSV is also up-regulated, as has been shown in selective immunoglobulin responses in other antigen stimulation settings [36] In patients with asthma the lack of correlation of age might be due to reaching a threshold of responses, where once attained, there is no clear feedback loop to regulate this response with time (e.g. age), or alternatively, it could be that it is contingent on the age of onset in addition to the time of antigen exposure and maternal transfer of Ab (blood or breast feeding), which may govern these collective responses [37]. However, subjects without asthma may possess a more linear response to antigen exposure, due to lack of hyper inflammations and potential dysregulation which exists in the asthmatic state [38,39] As such, the non asthmatic T/B cell profile or TH1/Th2 response may not have yet reached the threshold limits (since the virus specific Ab responses were actually lower than that of asthmatic patients). Or it could be in non asthma, there may be more responsiveness to potentiating virus specific antibody signals (e.g. cytokines) than that of the asthmatic patient. Such differences in cytokine responses in asthma compared with no asthma compared with atopic patients have been reported [39,40]. Others have reported excess Th2 –type cytokine immune responses, with deficient Th1 type responses, in RSV-infected infants [41], which may result in a more severe course of infection; Th1 type responses are required for efficient antiviral responses [41]. This study has limitations that should be considered in interpreting these findings, including: (1) modest sample size, and (2) recurrent wheezing in children is not always asthma, but might occur after viral infection. Despite these limitations, our study has carefully structured experimental research design and statistically significant findings. This study provides evidence of an association between RSV Abs in asthmatic children and IgE responses, and underscores the importance of IgE and viruses affecting the health of children with asthma. Future research efforts are necessary to develop new and effective therapeutics to prevent RSV disease, as well as testing new immunological biomarkers. Disclosure The authors declare no competing financial interest to disclose. Conflict of interest The authors declare no conflict of interest to disclose.
524
T.A. Smith-Norowitz et al. / Human Immunology 76 (2015) 519–524
Acknowledgments We thank Kevin B. Norowitz, M.D., (SUNY Downstate Medical Center, Department of Pediatrics) for helpful comments and thoughtful discussion. This study was funded by a NY State Divisional Grant/Pilot Project Grant. References [1] Everard ML, Milner AD. The respiratory syncytial virus and its role in acute bronchiolitis. Eur. J. Pediatr. 1992;151:638–51. [2] Hortal M, Russi JC, Arbiza JR, Canepa E, Chiparelli H, Illaramendy A. Identification of viruses in a study acute respiratory tract infection in children in Uruguay. Rev. Infect. Dis. 1990;12(Suppl. 8):S995–7. [3] Falsey AR, Hennessey PA, Formica MA, Cox C, Walsh EE. Respiratory syncytial virus infection in elderly and high risk adults. N. Engl. J. Med. 2005;352: 1749–59. [4] McIntosh K, Fishaut JM. Immunopathologic mechanisms in lower respiratory tract disease of infants due to respiratory syncytial virus. Prog. Med. Virol. 1980;26:94–118. [5] Bui RH, Molinaro GA, Kettering JD, Heiner DC, Imagawa DT, St Geme Jr JW. Virus-specific IgE and IgG4 antibodies in serum of children infected with respiratory syncytial virus. J. Pediatr. 1987;110:87–90. [6] Welliver RC, Sun M, Hildreth SW, Arumugham R, Ogra PL. Respiratory syncytial virus-specific antibody responses in immunoglobulin A and E isotypes to the F and G proteins and to intact virus after natural infection. J. Clin. Microbiol. 1989;27:295–9. [7] Bardin PG, Johnston SL, Pattermore PK. Viruses as precipitants of asthma symptoms. II. Physiology and mechanisms. Clin. Exp. Allergy 1992;22:809–22. [8] Rabatic S, Gagro A, Lokar-Kolbas R, Krsulovic-Hresic V, Vrtar Z, Popow-Kraupp T, et al. Increase in CD23+ B cells in infants with bronchiolitis is accompanied by appearance of IgE and IgG4 antibodies specific for respiratory syncytial virus. J. Infect. Dis. 1997;175:32–7. [9] Toms GL, Quinn R, Robinson JW. Undetectable IgE responses after respiratory syncytial virus infection. Arch. Dis. Child. 1996;74:126–30. [10] Pattenmore PK, Johnston SL, Bardin PG. Viruses as precipitants of asthma symptoms, I epidemiology. Clin. Exp. Allergy 1992;22:325–36. [11] Blanken MO, Rovers MM, Molenaar JM, Winkler-Seinstra PL, Meijer A, Kimpen JL, et al. Respiratory syncytial virus and recurrent wheeze in healthy preterm infants. N. Engl. J. Med. 2013;368:1791–9. [12] Zomer-Kooijker K, van der Ent CK, Ermers MJ, Uiterwaal CS, Rovers MM, Bont LJ, RSV Corticosteroid Study Group. Increased risk of wheeze and decreased lung function after respiratory syncytial virus infection. PLoS One 2014;9(1):e87162. http://dx.doi.org/10.1371/journal.pone.0087162. [13] Sigurs N, Aljassim F, Kjellman B, Robinson PD, Sigurbergsson F, Bjarnason R, Gustafsson PM. Asthma and allergy patterns over 18 years after severe RSV bronchiolitis in the first year of life. Thorax 2010;65:1045–52. [14] Smyth RL, Openshaw PJ. Bronchiolitis. Lancet 2006;368:312–22. [15] Gelfand EW. Pediatric asthma: a different disease. Proc. Am. Thorac. Soc. 2009;6:278–82. [16] Walton RP, Johnston SL. Role of respiratory viral infections in the development of atopic conditions. Curr. Opin. Allergy Clin. Immunol. 2008;8:150–3. [17] Cowen MK, Wakefield DB, Cloutier MM. Classifying asthma severity: objective versus subjective measures. J. Asthma 2007;44:711–5. [18] EPR-3, Expert panel report 3: guidelines for the diagnosis and management of asthma (EPR-3 2007), U.S. Department of Health and Human Services; National Institutes of Health; National Heart, Lung, and Blood Institute; National Asthma Education and Prevention Program, 2007; 40–3. http://www. nhlbi.nih.gov/guidelines/asthma/asthgdln.pdf. [19] Russi JS, Delfraro A, Borthagaray MD, Velazquez B, Barreno G, Hortal M. Evaluation of immunoglobulin E-specific antibodies and viral antigens in nasopharyngeal secretions of children with respiratory syncytial virus infection. J. Clin. Microbiol. 1993;31:819–23. [20] De Alcaron A, Walsh EE, Carper HT, La Russa JB, Evans BA, Rakes GP, et al. Detection of IgA and IgG but not IgE antibody to respiratory syncytial virus in nasal washes and sera from infants with wheezing. J. Pediatr. 2001;138:311–7.
[21] Welliver RC, Duffy L. The relationship of RSV-specific immunoglobulin E antibody responses in infancy, recurrent wheezing, and pulmonary function at age 7–8 years. Pediatr. Pulmonol. 1993;15:19–27. [22] Welliver RC, Sun M, Rinaldo D, Ogra PL. Predictive value of respiratory syncytial virus-specific IgE responses for recurrent wheezing following bronchiolitis. J. Pediatr. 1986;109:776–80. [23] Kwon J-M, Shim JW, Kim DS, Jung HL, Park MS, Shim JY. Prevalence of respiratory viral infection in children hospitalized for acute lower respiratory tract diseases, and association of rhinovirus and influenza virus with asthma exacerbations. Korean J. Pediatr. 2014;57:29–34. [24] Kumar RK, Hitchins MP, Foster PS. Epigenetic changes in childhood asthma. Dis. Model. Mech. 2009;2:549–53. [25] Gilmour J, Lavender P. Control of IL-4 expression in T helper 1 and 2 cells. Immunology 2008;124:437–44. [26] Weidinger S, Willis-Owen SA, Kamatani Y, Baurecht H, Morar N, Liang L, et al. A genome-wide association study of atopic dermatitis identifies loci with overlapping effects on asthma and psoriasis. Hum. Mol. Genet. 2013;22(23): 4841–56. [27] Viterbo D, Callender GE, DiMaio T, Mueller CM, Smith-Norowitz T, Zenilman ME, et al. Administration of anti-Reg I and anti PAP II antibodies worsens pancreatitis. JOP 2009;10:15–23. [28] Alonso MG, Caballero ML, Umpierrez A, Lluch-Bernal M, Knaute T, RodríguezPérez R. Relationships between T-cell and IgE/IgG4 epitopes of the Anisakis simplex major allergen Ani s 1. Clin. Exp. Allergy 2014. http://dx.doi.org/ 10.1111/cea.12474 [Epub ahead of print]; PMID: 25495594. [29] Quezada A, Norambuena X, Inostroza J, Rodríguez J. Specific antibody deficiency with normal immunoglobulin concentration in children with recurrent respiratory infections. Allergol. Immunopathol. (Madr.) 2014. http://dx.doi.org/10.1016/j.aller.2014.07.009. pii: S0301-0546(14)00162-1. [Epub ahead of print] PMID: 25498324. [30] Smith-Norowitz TA, Drew H, Norowitz HM, Bluth EF, Durkin HG, Bluth MH. Detection of IgE anti parvovirus antibodies in human breast milk. Ann. Clin. Lab. Sci. 2008;38:168–73. [31] Smith-Norowitz TA, Josekutty J, Lev-Tov H, Norowitz KB, Kohlhoff SA, Silverberg JI, et al. Long term persistence of IgE anti-Varicella Zoster Virus in pediatric and adult serum post chicken pox infection and after vaccination with Varicella Virus vaccine. Int. J. Biomed. Sci. 2009:100–5. [32] Smith-Norowitz TA, Josekutty J, Lev-Tov H, Kohlhoff S, Norowitz KB, Silverberg JI, et al. IgE anti-Varicella Zoster virus and other immune responses before, during, and after shingles. Ann. Clin. Lab. Sci. 2009;39:43–50. [33] Smith-Norowitz TA, Wong D, Kusonruksa M, Norowitz KB, Joks R, Durkin HG, et al. Long term persistence of IgE anti-influenza virus antibodies in pediatric and adult serum post vaccination with influenza virus vaccine. Int. J. Med. Sci. 2011;8:239–44. [34] Secord EA, Kleiner GI, Auci DL, Smith-Norowitz T, Chice S, Finkielstein A, et al. IgE against HIV proteins in clinically healthy children with HIV disease. J. Allergy Clin. Immunol. 1996;98:979–84. [35] Pellegrino MG, Bluth MH, Smith-Norowitz TA, Fikrig S, Volsky DJ, Moallem H, et al. HIV type 1-specific IgE in serum of long-term surviving children inhibits HIV type 1 production in vitro. AIDS Res. Hum. Retroviruses 2002; 18:363–72. [36] Fu SI, Pierre J, Smith-Norowitz TA, Hagler M, Bowner W, Pincus MR, et al. Immunuoglobulin E antibodies from pancreatic cancer patients mediate antibody-dependent cell-mediated cytotoxicity against pancreatic cancer cells. Clin. Exp. Immunol. 2008;153:401–9. [37] Dias AS, Gerber PF, Araujo AS, Auler PA, Gallinari GC, Lobato ZI. Lack of antibody protection against Porcine circovirus 2 and Porcine parvovirus in naturally infected dams and their offspring. Res. Vet. Sci. 2013;94:341–5. [38] Djukanovic R. Asthma: a disease of inflammation and repair. J. Allergy Clin. Immunol. 2000;105:S522–6. [39] Pala P, Message SD, Johnston SL, Openshaw PJM. Increased aeroallergenspecific interleukin-4-producing T cells in asthmatic adults. Clin. Exp. Allergy 2002;32:1739–44. [40] Tang C, Rolland IM, Ward C, Bish R, Thien F, Walters EH. Seasonal comparison of cytokine profiles in astopic asthmatics and atopic non asthmatics. Am. J. Respir. Crit. Care Med. 1996;154:1615–22. [41] Legg JP, Hussain IR, Warner JA, Johnston SL, Warner JO. Type 1 and type 2 cytokine imbalance in acute respiratory syncytial virus bronchiolitis. Am. J. Respir. Crit. Care Med. 2003;168:633–9.