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Association between TNF-α and TGF-β genotypes in infants and parental history of allergic rhinitis and asthma
Association Between TNF-␣ and TGF- Genotypes in Infants and Parental History of Allergic Rhinitis and Asthma Deborah A. Gentile, William J. Doyle, Adriana Zeevi, Judith Howe-Adams, Jordan Trecki, and David P. Skoner ABSTRACT: The development and expression of allergic rhinitis and asthma may be influenced by the elaboration of specific cytokines. Cytokine genotypes moderate illness severity in a variety of inflammatory disorders. Cytokine genotyping was performed on 124 infants (85% white, 57% male) to determine whether specific cytokine genotypes are associated with a parental history of allergic rhinitis and/or asthma. DNA was extracted from buccal brushings and assayed for tumor necrosis factor alpha (TNF-␣), interferon gamma (IFN-␥), interleukin (IL)-6, IL-10, and transforming growth factor (TGF)-1 genotypes using polymerase chain reaction–sequence specific primer technology. Outcomes consisted of parental history of allergy and asthma, and results were evaluated by logistic regression. TNF-␣ and TGF- genotypes were related to maternal and/or paternal history of allergic rhinitis and asthma, respectively. The frequencies of the genotype associated with high production of TNF-␣ were 41% versus 18% in infants with and without a parental
INTRODUCTION Allergic rhinitis and asthma are extremely common in pediatric and adult populations and have been associated with an alarming increase in prevalence, morbidity, and mortality [1, 2]. Additionally, these diseases are associated with a variety of complications and comorbid conditions, including otitis media, sinusitis, and pneumonia. The burden to society in terms of direct and indirect costs, and the impact on quality of life are quite substantial. Exposure to various environmental factors in-
Department of Pediatrics, Allegheny General Hospital (D.A.G., J.T., D.P.S., J.H.-A.), and Departments of Otolaryngology (W.J.D.) and Pathology (A.Z.), University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. Address reprint requests to: Dr. Deborah A. Gentile, Allegheny General Hospital, 320 E North Avenue, Pittsburgh, PA 15212; Tel: (412) 3596645; Fax: (412) 359-6640; E-mail: [email protected]. Received October 6, 2003; revised January 21, 2004; accepted January 23, 2004. Human Immunology 65, 347–351 (2004) © American Society for Histocompatibility and Immunogenetics, 2004 Published by Elsevier Inc.
history of allergic rhinitis, respectively (p ⬍ 0.01). The frequencies of the genotype associated with low production of TGF-1 were 14% versus 1% in infants with and without a parental history of asthma, respectively (p ⬍ 0.01). There were no associations between IFN-␥, IL-6, and IL-10 genotypes and any of the outcome parameters. These results suggest a role for TNF-␣ and TGF-1 genotypes in the pathogenesis of allergic rhinitis and asthma, respectively. If confirmed by future studies, cytokine genotyping may be a useful tool for identifying at-risk infants who may benefit from the selective use of preventative and/or early intervention treatments for these disorders. Human Immunology 65, 347–351 (2004). © American Society for Histocompatibility and Immunogenetics, 2004. Published by Elsevier Inc. KEYWORDS: cytokine genotypes; TNF-␣; TGF-1; asthma; allergic rhinitis
cluding allergens, tobacco smoke, and certain respiratory viruses are well-established risk factors for the development of allergic rhinitis and asthma [3–5]. However, the host factors that moderate these risks have not been elucidated. Results from previous studies demonstrate that a variety of pro- and anti-inflammatory cytokines are involved in the pathogenesis and expression of allergic rhinitis and asthma [6 – 8]. For example, tumor necrosis factor alpha (TNF-␣) is a proinflammatory cytokine that is often elevated in these disorders. The role of transforming growth factor (TGF)-1 is more complex and involves a dual role as both an anti-inflammatory and a profibrotic cytokine. Indeed, elevations of TGF-1 are associated with both dampened inflammation and enhanced remodeling of the airway in these disorders. In that regard, polymorphisms in the regulator regions of genes for production of these and other cytokines have 0198-8859/04/$–see front matter doi:10.1016/j.humimm.2004.01.014
348
been reported [9 –14], and specific cytokine genotypes have been related to the risk for illness severity in a variety of infectious and inflammatory diseases [15–20]. As part of an ongoing study examining the association between cytokine genotypes and the severity of illness and incidence of complications during acute infection with respiratory syncytial virus (RSV), 124 infants were genotyped for gene polymorphisms related to the level of interferon gamma (IFN-␥), interleukin (IL)-6, IL-10, TNF-␣, and TGF-1 [17]. Also, parental histories of allergic rhinitis and/or asthma were obtained. Previously, our investigative group reported that illness severity during RSV infection was associated with the genotype for IL-6 production and that the frequency of otitis media was associated with the genotype for IFN-␥ production [17]. In this report, we explored the relationship between infant cytokine genotypes and parental history of allergic rhinitis and/or asthma. PATIENTS AND METHODS Subjects All infant subjects were enrolled as part of an ongoing study examining the association between cytokine genotypes and severity of illness during RSV infection [17]. This study was approved by the Human Rights Committee of Children’s Hospital of Pittsburgh, and written informed consent was obtained from parents before screening and enrollment of the infants. Infants ⬍6 months of age were recruited from Children’s Hospital of Pittsburgh. Exclusion criteria included (1) birth prematurity (⬍36 weeks’ gestation), (2) use of oxygen and/or a ventilator beyond 6 hours after birth, (3) presence of major congenital anomalies, and (4) and presence of any congenital disorders of the chest, heart, or lungs. For comparative purposes, genotyping results from the infants were compared with those obtained from a large cohort of randomly selected adults recruited from the Pittsburgh area and enrolled in clinical studies of the common cold. Assessments of allergic rhinitis and asthma were not performed in those subjects. Protocol At enrollment, a detailed medical history and physical examination were completed, and a buccal brushing was obtained for DNA extraction and determination of cytokine genotypes. Race and parental history of allergic rhinitis and/or asthma were recorded. A buccal brushing was done by gently brushing each side of the buccal mucosa with a small nylon brush, and epithelial cells were immediately suspended in 1 ml of sterile saline and stored at ⫺20°C. For genotyping, buccal brushing samples were thawed and DNA was extracted with a commercially available
D.A. Gentile et al.
kit (QIAamp Mini Kit, Quiagen Inc., Valencia, CA) and protocols supplied by the manufacturer. Cytokine genotypes were determined using a commercially available kit (One Lambda Inc., Canoga Park, CA) that is routinely used for clinical purposes in organ transplantation, and provides sequence specific oligonucleotide primers for polymerase chain reaction (PCR) amplification of selected cytokine alleles including TNF-␣ (⫺308 G/A), TGF-1 (C/T codon 10, C/G codon 25), IL-10 (⫺1082 G/A, ⫺819 T/C, ⫺592 A/C), IL-6 (⫺174 G/C), and IFN-␥ (⫹874 A/T) [16, 17]. In brief, preoptimized primers were presented (dried) in different wells of a 96-well 0.2-ml thin-walled tube tray for PCR; DNA samples, recombinant Taq polymerase, and specially formulated dNTP buffer mix were added; PCR was performed with a Perkin-Elmer 9600 Thermocycler, and the presence or absence of PCR amplification was detected by gel electrophoresis and visualized by ethidium bromide staining. The DNA extractions and PCR amplifications were performed by a technician blinded to the clinical results. Many, but not all, studies have previously reported associations between these alleles and phenotypes that correspond to the expression levels of the cytokines [10 –14, 21]. Statistical Analysis Study outcomes consisted of parental history of allergic rhinitis and/or asthma. Predictor variables consisted of the genotypes for IL-6, IL-10, TNF-␣, INF-␥, and TGF1. Race was considered as a standard control variable. The effects of cytokine genotypes on the frequencies of parental history of allergic rhinitis and/or asthma were evaluated for significance by a step-down logistic regression method with predictor variables retained in the model if significant at alpha p ⬍ 0.05. The effects of cytokine genotypes on the score and time variables were evaluated by a logistic regression method. Predictor variables with significant effects were reanalyzed with inclusion of the control variables using those methods. Significance of model effects was defined at alpha p ⬍ 0.05. All statistical procedures were run using the NCSS 2000 statistical package (Kaysville, UT). RESULTS A total of 124 infants (15% black and 85% white; 43% female and 57% male) were enrolled. Table 1 shows the percentage distributions of cytokine genotypes for the 124 enrolled infants and for 107 randomly selected adults recruited from the Pittsburgh area. A comparison of these distributions between the adult and infant populations showed no significant differences. The frequencies of maternal, paternal, and maternal and/or paternal allergic rhinitis were 15%, 15%, and 28%, respectively.
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TABLE 1 Distribution of cytokine gene polymorphisms in study infants and randomly selected adults Infants (n ⫽ 124) Low Intermediate High Adults (n ⫽ 107) Low Intermediate High
TNF-␣
TGF-1
IL-10
IL-6
IFN-␥
74%
4% 25% 71%
40% 42% 18%
16%
31% 45% 24%
5% 25% 70%
39% 47% 14%
26% 74% 26%
84% 16% 84%
29% 50% 21%
The frequencies of maternal, paternal, and maternal and/or paternal asthma were 34%, 22%, and 46%, respectively. There were significant associations between TNF-␣ and TGF-1 genotypes and parental history of allergic rhinitis and asthma, respectively. Specifically, the genotypes associated with high TNF-␣ production (⫺308A/A and ⫺308G/A) were identified at a significantly increased frequency (p ⬍ 0.01) in infants with, as compared with those without, a history of parental allergic rhinitis (Figure 1). Conversely, the genotypes associated with low TGF-1 production (codons 10, 25 C/C-G/C, C/C-C/C, T/T-C/C, T/C-C/C) were identified at a significantly increased frequency (p ⬍ 0.01) in infants with, as compared with those without, a history of parental asthma (Figure 2). There were no significant interactions between TNF-␥ and TGF-1 genotypes and the outcomes. There were no associations between IFN-␥, IL-6, and IL-10 genotypes and parental history of allergic rhinitis or asthma. DISCUSSION Local and systemic levels of proinflammatory cytokines are elevated in patients with allergic diseases, including
FIGURE 1 Distribution of the gene polymorphisms associated with high TNF-␣ production in infants with (closed bars) and without (open bars) a parental history of allergic rhinitis (p ⬍ 0.01 for those with vs. those without a history of allergic rhinitis in mother, father, or either).
FIGURE 2 Distribution of gene polymorphisms associated with low TGF-1 production in infants with (closed bars) or without (open bars) a parental history of asthma (p ⬍ 0.01 for those with vs. those without a history of asthma in mother, father, or either).
rhinitis and/or asthma [6 – 8]. For example, TNF-␣ is recognized as a proinflammatory host alert cytokine that is synthesized early during inflammation, and elevations of this cytokine have been reported in patients with allergic rhinitis [6]. Conversely, TGF-1 is considered to be an anti-inflammatory cytokine, as evidenced by an early study that demonstrated an association between low production of this protein and increased severity of asthma [8]. These observations have been confirmed in animal studies of these disorders. Specifically, murine models have been used to demonstrate increased levels of serum TNF-␣ protein after allergic sensitization [22], as well as the beneficial effects of TGF-1 on allergeninduced airway hyperreactivity and inflammation [23, 24]. Collectively, these observations strongly suggest that an imbalance in the levels of these cytokines contributes to the pathogenesis and expression of allergic rhinitis and asthma. One factor controlling the level of cytokine expression is cytokine genotype. Specifically, polymorphisms in the regulatory regions of cytokine genes may affect the message for and production of cytokine genes in response to various stimuli [10 –14]. Also, an individual’s genotype for certain cytokines was shown to be prognostic for the susceptibility to immunologic and inflammatory diseases [15–20]. The present study explored the relationship between cytokine genotypes in infants and parental history of allergic rhinitis and/or asthma. The results showed that the allele frequencies of each of the examined cytokine genotypes were similar in the enrolled infants and the general population. However, the genotypes for high production of TNF-␣ and low production of TGF-1 were associated with an increased frequency of parental allergic rhinitis and asthma, respectively. These results are consistent with those reported by several other groups examining the association between genotypes for TNF-␣ and TGF-1 production and incidence and severity of allergic diseases in various populations [25–28].
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Although providing an intriguing set of relationships between cytokine genotypes and parental history of allergic rhinitis and/or asthma, several study limitations confine these interpretations to hypotheses for future testing. First, the panel of cytokine genotypes assessed was very selective and may not have included other important genotypes, which influence the pathogenesis and expression of allergic rhinitis and/or asthma. In fact, recent studies have reported associations between other cytokine genotypes, including T helper type 2 (Th2) cytokine genotypes, and these diseases [29, 30]. Also, it is conceivable that polymorphisms for other immunomodulatory molecules, including cytokine receptors, adhesion molecules, and costimulatory molecules, may play an important role. Second, although the present study documented associations between specific cytokine genotypes in infants and parental history of allergic rhinitis and/or asthma, it did not determine whether cytokine genotype was predictive of phenotypic expression. Some, but not all, studies have reported associations between cytokine genotype, mRNA expression, and protein levels [10 –14, 21]. Additionally, a variety of transcriptional, translational, and posttranslational mechanisms can influence cytokine production [13, 14]. Third, cytokine genotyping was not performed in the parents and infants were not followed longitudinally to assess for the development of allergic rhinitis and/or asthma, and this prevented a direct comparison between their genotypes and frequency of reported disease. Fourth, the assignment of disease was based on parental report and was not standardized by allergy skin testing, serum Immunoglobulin E levels, lung function testing or assessment of lower airway hyperreactivity. Other limitations are related to the very low number of subjects with the genotype associated with low TGF-1 production, the racial distribution of subjects, and the resultant possibility of a type I error. Our investigative team plans to incorporate prospective, longitudinal follow-up of infants to assess the development of allergic rhinitis and/or asthma, parental genotyping, standardized assessments of disease, and assessments of the relationship between genotypes and phenotypes into future studies. If confirmed, the reported associations may define subgroups of infants at risk for the development of allergic rhinitis and/or asthma. Once identified, such individuals may benefit from the selective use of preventative and/or early intervention treatments for these diseases. [2] ACKNOWLEDGMENT
We acknowledge Gina Phillape, for performing the cytokine genotyping.
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REFERENCES 1. Centers for Disease Control: Self-reported asthma prevalence and control among adults—United States, 2001. MMWR Morb Mortal Wkly Rep 52:1, 2003. 2. Brown P, Mayer B, Zavestoski S, Luebke T, Mandelbaum J, McCormick S: The health politics of asthma: environmental justice and collective illness experience in the United States. Soc Sci Med 57:453, 2003. 3. Kurukulaaratchy RJ, Matthews S, Waterhouse L, Arshad SH: Factors influencing symptom expression in children with bronchial hyperresponsiveness at 10 years of age. J Allergy Clin Immunol 112:311, 2003. 4. Belanger K, Beckett W, Triche E, Bracken MB, Holford T, Ren P, McSharry JE, Gold DR, Platts-Mills TA, Leaderer BP: Symptoms of wheeze and persistent cough in the first year of life: associations with indoor allergens, air contaminants, and maternal history of asthma. Am J Epidemiol 158:195, 2003. 5. Gern JE, Lemanske RF: Infectious triggers of pediatric asthma. Pediatr Clin North Am 50:555, 2003. 6. Riccio AM, Tosca MA, Cosentino C, Pallestrini E, Ameli F, Canonica GW, Ciprandi G: Cytokine pattern in allergic and non-allergic chronic rhinosinusitis in asthmatic children. Clin Exp Allergy 32:422, 2002. 7. Liebhart J, Cembrzynska-Nowak M, Bienkowska M, Liebhart E, Dobek R, Zaczynska E, Panaszek B, Obojski A, Malolepszy J: Relevance of the selected cytokine release to the exacerbation of bronchial asthma from airway mycotic infection. J Investig Allergol Clin Immunol 12:182, 2002. 8. Jutel M, Akdis M, Budak F, Aebischer-Casaulta C, Wrzyzcz M, Blaser K, Akdis CA: IL-10 and TGF- cooperate in the regulatory T cell response to mucosal allergens in normal immunity and specific immunotherapy. Eur J Immunol 33:1205, 2003. 9. Fishman D, Faulds G, Jeffrey R, Mohamed-Ali V, Yudkin JS, Humphries S, Woo P: The effect of novel polymorphisms in the IL-6 gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic-onset juvenile chronic arthritis. J Clin Invest 102:1369, 1998. 10. Schaaf BM, Boehmke F, Esnaashar H, Seitzer U, Kothe H, Maass M, Zabel P, Dalhoff K: Pneumococcal septic shock is associated with the interleukin-10-1082 gene promoter polymorphism. Am J Respir Crit Care Med 168:476, 2003. 11. Hoffmann SC, Stanley EM, Darrin Cox E, Craighead N, DiMercurio BS, Koziol DE, Harlan DM, Kirk AD, Blair PJ: Association of cytokine polymorphic inheritance and in vitro cytokine production in anti-CD3/CD28-stimulated peripheral blood lymphocytes. Transplantation 72: 1444, 2001. 12. Temple SE, Lim E, Cheong KY, Almeida Ca, Price P, Ardlie KG, Waterer GW. Alleles carried at positions ⫺819 and ⫺592 of the IL10 promoter affect transcription
Cytokine Genotypes and Asthma/Allergy
13.
14.
15.
16.
17.
18.
19.
20.
21.
following stimulation of peripheral blood cells with Streptococcus pneumonia. 55:629, 2003. Louis E, Franchimont D, Piron A, Gevaert Y, SchaafLafontaine N, Roland S, Mahieu M, De Groote D, Louis R, Belaiche J: Tumor necrosis factor (TNF) gene polymorphism influences TNF-alpha production in LPS-stimulated whole blood cell culture in healthy humans. Clin Exp Immunol 113:401, 1998. Awad MR, El-Gamel A, Hasleton P, Turner DM, Sinnott PJ, Hutchinson IV: Genotypic variation in the transforming growth factor-beta 1 gene: association with transforming growth factor-beta 1 production, fibrotic lung disease, and graft fibrosis after lung transplantation. Transplantation 66:1014, 1998. Simhan HN, Krohn MA, Zeevi A, Daftary A, Harger G, Caritis SN: TNF-␣ promoter gene polymorphism ⫺308 and chorioamnionitis. Obstet Gynecol 102:162, 2003. Gentile DA, Doyle WJ, Zeevi A, Piltcher O, Skoner DP: Cytokine gene polymorphisms moderate responses to respiratory syncytial virus in adults. Hum Immunol 64:93, 2003. Gentile DA, Doyle WJ, Zeevi A, Howe-Adams J, Kapadia S, Trecki J, Skoner DP: Cytokine gene polymorphisms moderate illness severity in infants with respiratory syncytial virus infection. Hum Immunol 64:338, 2003. Mira J, Cariou A, Grall F, Delclaux C: Association of TNF2, a TNF-␣ promoter polymorphism, with septic shock and susceptibility to mortality. JAMA 282:561, 1999. Sankaran D, Asderakis A, Ashraf S, Roberts I, Short CD, Dyer PA, Sinnott PJ, Hutchinson IV: Cytokine gene polymorphisms predict acute graft rejection following renal transplantation. Kidney Int 56:281, 1999. Awad RA, Webber S, Boyle G, Sturchioc C, Ahmed M, Martell J, Law Y, Miller SA, Bowman P, Gribar S, Pigula F, Mazariegos G, Griffith BP, Zeevi A: The effect of cytokine gene polymorphisms on pediatric heart allograft outcome. J Heart Lung Transplant 20:265, 2001. Cartwright NH, Keen LJ, Demaine AG, Hurlock NJ, McGonigle RJ, Rowe PA, Shaw JF, Szydlo RM, Kaminski
351
22.
23.
24.
25.
26.
27.
28.
29.
30.
ER: A study of cytokine gene polymorphisms and protein secretion in renal transplantation. Transpl Immunol 8:237, 2001. Ayoub M, Lallouette P, Sutterlin BW, Bessler WG, Huber M, Mittenbuhler: Modulation of the Th1/Th2 bias by an immunoglobulin histamine complex in the ovalbumin allergy mouse model. Int Immunopharmacol 3:523, 2003. Schramm C, Herz U, Podlech J, Protschka M, Finotto S, Reddehase MJ, Kohler H, Galle PR, Lohse AW, Blessing M: TGF-beta regulates airway responses via T cells. J Immunol 170:1313, 2003. Hansen G, McIntire JJ, Yeung VP, Berry G, Thorbecke GJ, Chen L, DeKruyff RH, Umetsu DT: CD4(⫹) T helper cells engineered to produce latent TGF-beta1 reverse allergen-induced airway hyperreactivity and inflammation. J Clin Invest 105:61, 2000. Pulleyn LJ, Newton R, Adcock IM, Barnes PJ: TGF-1 allele association with asthma severity. Hum Genet 109: 623, 2001. Hobbs K, Negri J, Klinnert M, Rosenwasser LJ, Borish L: IL-10 and TGF- promotor polymorphisms in allergies and asthma. Am J Respir Crit Care Med 158:1958, 1998. Zhu S, Chan-Yeung M, Becker AB, Dimich-Ward H, Ferguson AC, Manfreda J, Watson WT, Pare PD, Sandford AJ: Polymorphisms of the IL-4, TNF-␣, and Fc epsilon RI genes and the risk of allergic disorders in at-risk infants. Am J Respir Crit Care Med 161:1655, 2002. Witte JS, Palmer LJ, O’Connor RD, Hopkins PJ, Hall JM: Relation between TNF polymorphism TNF-␣-308 and risk of asthma. Eur J Hum Genet 10:82, 2002. Beghe B, Barton S, Rorke S, Peng Q, Sayers I, Gaunt T, Keith TP, Clough JB, Holgate ST, Holloway JS: Polymorphisms in the IL-4 and IL-4 receptor alpha chain genes confer susceptibility to asthma and atopy in a Caucasian population. Clin Exp Allergy 33:1111, 2003. Cui T, Wu J, Pan S, Xie J: Polymorphisms in the IL-4 and IL-4R (alpha) genes and allergic asthma. Clin Chem Lab Med 41:888, 2003.
INTRODUCTION Allergic rhinitis and asthma are extremely common in pediatric and adult populations and have been associated with an alarming increase in prevalence, morbidity, and mortality [1, 2]. Additionally, these diseases are associated with a variety of complications and comorbid conditions, including otitis media, sinusitis, and pneumonia. The burden to society in terms of direct and indirect costs, and the impact on quality of life are quite substantial. Exposure to various environmental factors in-
Department of Pediatrics, Allegheny General Hospital (D.A.G., J.T., D.P.S., J.H.-A.), and Departments of Otolaryngology (W.J.D.) and Pathology (A.Z.), University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. Address reprint requests to: Dr. Deborah A. Gentile, Allegheny General Hospital, 320 E North Avenue, Pittsburgh, PA 15212; Tel: (412) 3596645; Fax: (412) 359-6640; E-mail: [email protected]. Received October 6, 2003; revised January 21, 2004; accepted January 23, 2004. Human Immunology 65, 347–351 (2004) © American Society for Histocompatibility and Immunogenetics, 2004 Published by Elsevier Inc.
history of allergic rhinitis, respectively (p ⬍ 0.01). The frequencies of the genotype associated with low production of TGF-1 were 14% versus 1% in infants with and without a parental history of asthma, respectively (p ⬍ 0.01). There were no associations between IFN-␥, IL-6, and IL-10 genotypes and any of the outcome parameters. These results suggest a role for TNF-␣ and TGF-1 genotypes in the pathogenesis of allergic rhinitis and asthma, respectively. If confirmed by future studies, cytokine genotyping may be a useful tool for identifying at-risk infants who may benefit from the selective use of preventative and/or early intervention treatments for these disorders. Human Immunology 65, 347–351 (2004). © American Society for Histocompatibility and Immunogenetics, 2004. Published by Elsevier Inc. KEYWORDS: cytokine genotypes; TNF-␣; TGF-1; asthma; allergic rhinitis
cluding allergens, tobacco smoke, and certain respiratory viruses are well-established risk factors for the development of allergic rhinitis and asthma [3–5]. However, the host factors that moderate these risks have not been elucidated. Results from previous studies demonstrate that a variety of pro- and anti-inflammatory cytokines are involved in the pathogenesis and expression of allergic rhinitis and asthma [6 – 8]. For example, tumor necrosis factor alpha (TNF-␣) is a proinflammatory cytokine that is often elevated in these disorders. The role of transforming growth factor (TGF)-1 is more complex and involves a dual role as both an anti-inflammatory and a profibrotic cytokine. Indeed, elevations of TGF-1 are associated with both dampened inflammation and enhanced remodeling of the airway in these disorders. In that regard, polymorphisms in the regulator regions of genes for production of these and other cytokines have 0198-8859/04/$–see front matter doi:10.1016/j.humimm.2004.01.014
348
been reported [9 –14], and specific cytokine genotypes have been related to the risk for illness severity in a variety of infectious and inflammatory diseases [15–20]. As part of an ongoing study examining the association between cytokine genotypes and the severity of illness and incidence of complications during acute infection with respiratory syncytial virus (RSV), 124 infants were genotyped for gene polymorphisms related to the level of interferon gamma (IFN-␥), interleukin (IL)-6, IL-10, TNF-␣, and TGF-1 [17]. Also, parental histories of allergic rhinitis and/or asthma were obtained. Previously, our investigative group reported that illness severity during RSV infection was associated with the genotype for IL-6 production and that the frequency of otitis media was associated with the genotype for IFN-␥ production [17]. In this report, we explored the relationship between infant cytokine genotypes and parental history of allergic rhinitis and/or asthma. PATIENTS AND METHODS Subjects All infant subjects were enrolled as part of an ongoing study examining the association between cytokine genotypes and severity of illness during RSV infection [17]. This study was approved by the Human Rights Committee of Children’s Hospital of Pittsburgh, and written informed consent was obtained from parents before screening and enrollment of the infants. Infants ⬍6 months of age were recruited from Children’s Hospital of Pittsburgh. Exclusion criteria included (1) birth prematurity (⬍36 weeks’ gestation), (2) use of oxygen and/or a ventilator beyond 6 hours after birth, (3) presence of major congenital anomalies, and (4) and presence of any congenital disorders of the chest, heart, or lungs. For comparative purposes, genotyping results from the infants were compared with those obtained from a large cohort of randomly selected adults recruited from the Pittsburgh area and enrolled in clinical studies of the common cold. Assessments of allergic rhinitis and asthma were not performed in those subjects. Protocol At enrollment, a detailed medical history and physical examination were completed, and a buccal brushing was obtained for DNA extraction and determination of cytokine genotypes. Race and parental history of allergic rhinitis and/or asthma were recorded. A buccal brushing was done by gently brushing each side of the buccal mucosa with a small nylon brush, and epithelial cells were immediately suspended in 1 ml of sterile saline and stored at ⫺20°C. For genotyping, buccal brushing samples were thawed and DNA was extracted with a commercially available
D.A. Gentile et al.
kit (QIAamp Mini Kit, Quiagen Inc., Valencia, CA) and protocols supplied by the manufacturer. Cytokine genotypes were determined using a commercially available kit (One Lambda Inc., Canoga Park, CA) that is routinely used for clinical purposes in organ transplantation, and provides sequence specific oligonucleotide primers for polymerase chain reaction (PCR) amplification of selected cytokine alleles including TNF-␣ (⫺308 G/A), TGF-1 (C/T codon 10, C/G codon 25), IL-10 (⫺1082 G/A, ⫺819 T/C, ⫺592 A/C), IL-6 (⫺174 G/C), and IFN-␥ (⫹874 A/T) [16, 17]. In brief, preoptimized primers were presented (dried) in different wells of a 96-well 0.2-ml thin-walled tube tray for PCR; DNA samples, recombinant Taq polymerase, and specially formulated dNTP buffer mix were added; PCR was performed with a Perkin-Elmer 9600 Thermocycler, and the presence or absence of PCR amplification was detected by gel electrophoresis and visualized by ethidium bromide staining. The DNA extractions and PCR amplifications were performed by a technician blinded to the clinical results. Many, but not all, studies have previously reported associations between these alleles and phenotypes that correspond to the expression levels of the cytokines [10 –14, 21]. Statistical Analysis Study outcomes consisted of parental history of allergic rhinitis and/or asthma. Predictor variables consisted of the genotypes for IL-6, IL-10, TNF-␣, INF-␥, and TGF1. Race was considered as a standard control variable. The effects of cytokine genotypes on the frequencies of parental history of allergic rhinitis and/or asthma were evaluated for significance by a step-down logistic regression method with predictor variables retained in the model if significant at alpha p ⬍ 0.05. The effects of cytokine genotypes on the score and time variables were evaluated by a logistic regression method. Predictor variables with significant effects were reanalyzed with inclusion of the control variables using those methods. Significance of model effects was defined at alpha p ⬍ 0.05. All statistical procedures were run using the NCSS 2000 statistical package (Kaysville, UT). RESULTS A total of 124 infants (15% black and 85% white; 43% female and 57% male) were enrolled. Table 1 shows the percentage distributions of cytokine genotypes for the 124 enrolled infants and for 107 randomly selected adults recruited from the Pittsburgh area. A comparison of these distributions between the adult and infant populations showed no significant differences. The frequencies of maternal, paternal, and maternal and/or paternal allergic rhinitis were 15%, 15%, and 28%, respectively.
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TABLE 1 Distribution of cytokine gene polymorphisms in study infants and randomly selected adults Infants (n ⫽ 124) Low Intermediate High Adults (n ⫽ 107) Low Intermediate High
TNF-␣
TGF-1
IL-10
IL-6
IFN-␥
74%
4% 25% 71%
40% 42% 18%
16%
31% 45% 24%
5% 25% 70%
39% 47% 14%
26% 74% 26%
84% 16% 84%
29% 50% 21%
The frequencies of maternal, paternal, and maternal and/or paternal asthma were 34%, 22%, and 46%, respectively. There were significant associations between TNF-␣ and TGF-1 genotypes and parental history of allergic rhinitis and asthma, respectively. Specifically, the genotypes associated with high TNF-␣ production (⫺308A/A and ⫺308G/A) were identified at a significantly increased frequency (p ⬍ 0.01) in infants with, as compared with those without, a history of parental allergic rhinitis (Figure 1). Conversely, the genotypes associated with low TGF-1 production (codons 10, 25 C/C-G/C, C/C-C/C, T/T-C/C, T/C-C/C) were identified at a significantly increased frequency (p ⬍ 0.01) in infants with, as compared with those without, a history of parental asthma (Figure 2). There were no significant interactions between TNF-␥ and TGF-1 genotypes and the outcomes. There were no associations between IFN-␥, IL-6, and IL-10 genotypes and parental history of allergic rhinitis or asthma. DISCUSSION Local and systemic levels of proinflammatory cytokines are elevated in patients with allergic diseases, including
FIGURE 1 Distribution of the gene polymorphisms associated with high TNF-␣ production in infants with (closed bars) and without (open bars) a parental history of allergic rhinitis (p ⬍ 0.01 for those with vs. those without a history of allergic rhinitis in mother, father, or either).
FIGURE 2 Distribution of gene polymorphisms associated with low TGF-1 production in infants with (closed bars) or without (open bars) a parental history of asthma (p ⬍ 0.01 for those with vs. those without a history of asthma in mother, father, or either).
rhinitis and/or asthma [6 – 8]. For example, TNF-␣ is recognized as a proinflammatory host alert cytokine that is synthesized early during inflammation, and elevations of this cytokine have been reported in patients with allergic rhinitis [6]. Conversely, TGF-1 is considered to be an anti-inflammatory cytokine, as evidenced by an early study that demonstrated an association between low production of this protein and increased severity of asthma [8]. These observations have been confirmed in animal studies of these disorders. Specifically, murine models have been used to demonstrate increased levels of serum TNF-␣ protein after allergic sensitization [22], as well as the beneficial effects of TGF-1 on allergeninduced airway hyperreactivity and inflammation [23, 24]. Collectively, these observations strongly suggest that an imbalance in the levels of these cytokines contributes to the pathogenesis and expression of allergic rhinitis and asthma. One factor controlling the level of cytokine expression is cytokine genotype. Specifically, polymorphisms in the regulatory regions of cytokine genes may affect the message for and production of cytokine genes in response to various stimuli [10 –14]. Also, an individual’s genotype for certain cytokines was shown to be prognostic for the susceptibility to immunologic and inflammatory diseases [15–20]. The present study explored the relationship between cytokine genotypes in infants and parental history of allergic rhinitis and/or asthma. The results showed that the allele frequencies of each of the examined cytokine genotypes were similar in the enrolled infants and the general population. However, the genotypes for high production of TNF-␣ and low production of TGF-1 were associated with an increased frequency of parental allergic rhinitis and asthma, respectively. These results are consistent with those reported by several other groups examining the association between genotypes for TNF-␣ and TGF-1 production and incidence and severity of allergic diseases in various populations [25–28].
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Although providing an intriguing set of relationships between cytokine genotypes and parental history of allergic rhinitis and/or asthma, several study limitations confine these interpretations to hypotheses for future testing. First, the panel of cytokine genotypes assessed was very selective and may not have included other important genotypes, which influence the pathogenesis and expression of allergic rhinitis and/or asthma. In fact, recent studies have reported associations between other cytokine genotypes, including T helper type 2 (Th2) cytokine genotypes, and these diseases [29, 30]. Also, it is conceivable that polymorphisms for other immunomodulatory molecules, including cytokine receptors, adhesion molecules, and costimulatory molecules, may play an important role. Second, although the present study documented associations between specific cytokine genotypes in infants and parental history of allergic rhinitis and/or asthma, it did not determine whether cytokine genotype was predictive of phenotypic expression. Some, but not all, studies have reported associations between cytokine genotype, mRNA expression, and protein levels [10 –14, 21]. Additionally, a variety of transcriptional, translational, and posttranslational mechanisms can influence cytokine production [13, 14]. Third, cytokine genotyping was not performed in the parents and infants were not followed longitudinally to assess for the development of allergic rhinitis and/or asthma, and this prevented a direct comparison between their genotypes and frequency of reported disease. Fourth, the assignment of disease was based on parental report and was not standardized by allergy skin testing, serum Immunoglobulin E levels, lung function testing or assessment of lower airway hyperreactivity. Other limitations are related to the very low number of subjects with the genotype associated with low TGF-1 production, the racial distribution of subjects, and the resultant possibility of a type I error. Our investigative team plans to incorporate prospective, longitudinal follow-up of infants to assess the development of allergic rhinitis and/or asthma, parental genotyping, standardized assessments of disease, and assessments of the relationship between genotypes and phenotypes into future studies. If confirmed, the reported associations may define subgroups of infants at risk for the development of allergic rhinitis and/or asthma. Once identified, such individuals may benefit from the selective use of preventative and/or early intervention treatments for these diseases. [2] ACKNOWLEDGMENT
We acknowledge Gina Phillape, for performing the cytokine genotyping.
D.A. Gentile et al.
REFERENCES 1. Centers for Disease Control: Self-reported asthma prevalence and control among adults—United States, 2001. MMWR Morb Mortal Wkly Rep 52:1, 2003. 2. Brown P, Mayer B, Zavestoski S, Luebke T, Mandelbaum J, McCormick S: The health politics of asthma: environmental justice and collective illness experience in the United States. Soc Sci Med 57:453, 2003. 3. Kurukulaaratchy RJ, Matthews S, Waterhouse L, Arshad SH: Factors influencing symptom expression in children with bronchial hyperresponsiveness at 10 years of age. J Allergy Clin Immunol 112:311, 2003. 4. Belanger K, Beckett W, Triche E, Bracken MB, Holford T, Ren P, McSharry JE, Gold DR, Platts-Mills TA, Leaderer BP: Symptoms of wheeze and persistent cough in the first year of life: associations with indoor allergens, air contaminants, and maternal history of asthma. Am J Epidemiol 158:195, 2003. 5. Gern JE, Lemanske RF: Infectious triggers of pediatric asthma. Pediatr Clin North Am 50:555, 2003. 6. Riccio AM, Tosca MA, Cosentino C, Pallestrini E, Ameli F, Canonica GW, Ciprandi G: Cytokine pattern in allergic and non-allergic chronic rhinosinusitis in asthmatic children. Clin Exp Allergy 32:422, 2002. 7. Liebhart J, Cembrzynska-Nowak M, Bienkowska M, Liebhart E, Dobek R, Zaczynska E, Panaszek B, Obojski A, Malolepszy J: Relevance of the selected cytokine release to the exacerbation of bronchial asthma from airway mycotic infection. J Investig Allergol Clin Immunol 12:182, 2002. 8. Jutel M, Akdis M, Budak F, Aebischer-Casaulta C, Wrzyzcz M, Blaser K, Akdis CA: IL-10 and TGF- cooperate in the regulatory T cell response to mucosal allergens in normal immunity and specific immunotherapy. Eur J Immunol 33:1205, 2003. 9. Fishman D, Faulds G, Jeffrey R, Mohamed-Ali V, Yudkin JS, Humphries S, Woo P: The effect of novel polymorphisms in the IL-6 gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic-onset juvenile chronic arthritis. J Clin Invest 102:1369, 1998. 10. Schaaf BM, Boehmke F, Esnaashar H, Seitzer U, Kothe H, Maass M, Zabel P, Dalhoff K: Pneumococcal septic shock is associated with the interleukin-10-1082 gene promoter polymorphism. Am J Respir Crit Care Med 168:476, 2003. 11. Hoffmann SC, Stanley EM, Darrin Cox E, Craighead N, DiMercurio BS, Koziol DE, Harlan DM, Kirk AD, Blair PJ: Association of cytokine polymorphic inheritance and in vitro cytokine production in anti-CD3/CD28-stimulated peripheral blood lymphocytes. Transplantation 72: 1444, 2001. 12. Temple SE, Lim E, Cheong KY, Almeida Ca, Price P, Ardlie KG, Waterer GW. Alleles carried at positions ⫺819 and ⫺592 of the IL10 promoter affect transcription
Cytokine Genotypes and Asthma/Allergy
13.
14.
15.
16.
17.
18.
19.
20.
21.
following stimulation of peripheral blood cells with Streptococcus pneumonia. 55:629, 2003. Louis E, Franchimont D, Piron A, Gevaert Y, SchaafLafontaine N, Roland S, Mahieu M, De Groote D, Louis R, Belaiche J: Tumor necrosis factor (TNF) gene polymorphism influences TNF-alpha production in LPS-stimulated whole blood cell culture in healthy humans. Clin Exp Immunol 113:401, 1998. Awad MR, El-Gamel A, Hasleton P, Turner DM, Sinnott PJ, Hutchinson IV: Genotypic variation in the transforming growth factor-beta 1 gene: association with transforming growth factor-beta 1 production, fibrotic lung disease, and graft fibrosis after lung transplantation. Transplantation 66:1014, 1998. Simhan HN, Krohn MA, Zeevi A, Daftary A, Harger G, Caritis SN: TNF-␣ promoter gene polymorphism ⫺308 and chorioamnionitis. Obstet Gynecol 102:162, 2003. Gentile DA, Doyle WJ, Zeevi A, Piltcher O, Skoner DP: Cytokine gene polymorphisms moderate responses to respiratory syncytial virus in adults. Hum Immunol 64:93, 2003. Gentile DA, Doyle WJ, Zeevi A, Howe-Adams J, Kapadia S, Trecki J, Skoner DP: Cytokine gene polymorphisms moderate illness severity in infants with respiratory syncytial virus infection. Hum Immunol 64:338, 2003. Mira J, Cariou A, Grall F, Delclaux C: Association of TNF2, a TNF-␣ promoter polymorphism, with septic shock and susceptibility to mortality. JAMA 282:561, 1999. Sankaran D, Asderakis A, Ashraf S, Roberts I, Short CD, Dyer PA, Sinnott PJ, Hutchinson IV: Cytokine gene polymorphisms predict acute graft rejection following renal transplantation. Kidney Int 56:281, 1999. Awad RA, Webber S, Boyle G, Sturchioc C, Ahmed M, Martell J, Law Y, Miller SA, Bowman P, Gribar S, Pigula F, Mazariegos G, Griffith BP, Zeevi A: The effect of cytokine gene polymorphisms on pediatric heart allograft outcome. J Heart Lung Transplant 20:265, 2001. Cartwright NH, Keen LJ, Demaine AG, Hurlock NJ, McGonigle RJ, Rowe PA, Shaw JF, Szydlo RM, Kaminski
351
22.
23.
24.
25.
26.
27.
28.
29.
30.
ER: A study of cytokine gene polymorphisms and protein secretion in renal transplantation. Transpl Immunol 8:237, 2001. Ayoub M, Lallouette P, Sutterlin BW, Bessler WG, Huber M, Mittenbuhler: Modulation of the Th1/Th2 bias by an immunoglobulin histamine complex in the ovalbumin allergy mouse model. Int Immunopharmacol 3:523, 2003. Schramm C, Herz U, Podlech J, Protschka M, Finotto S, Reddehase MJ, Kohler H, Galle PR, Lohse AW, Blessing M: TGF-beta regulates airway responses via T cells. J Immunol 170:1313, 2003. Hansen G, McIntire JJ, Yeung VP, Berry G, Thorbecke GJ, Chen L, DeKruyff RH, Umetsu DT: CD4(⫹) T helper cells engineered to produce latent TGF-beta1 reverse allergen-induced airway hyperreactivity and inflammation. J Clin Invest 105:61, 2000. Pulleyn LJ, Newton R, Adcock IM, Barnes PJ: TGF-1 allele association with asthma severity. Hum Genet 109: 623, 2001. Hobbs K, Negri J, Klinnert M, Rosenwasser LJ, Borish L: IL-10 and TGF- promotor polymorphisms in allergies and asthma. Am J Respir Crit Care Med 158:1958, 1998. Zhu S, Chan-Yeung M, Becker AB, Dimich-Ward H, Ferguson AC, Manfreda J, Watson WT, Pare PD, Sandford AJ: Polymorphisms of the IL-4, TNF-␣, and Fc epsilon RI genes and the risk of allergic disorders in at-risk infants. Am J Respir Crit Care Med 161:1655, 2002. Witte JS, Palmer LJ, O’Connor RD, Hopkins PJ, Hall JM: Relation between TNF polymorphism TNF-␣-308 and risk of asthma. Eur J Hum Genet 10:82, 2002. Beghe B, Barton S, Rorke S, Peng Q, Sayers I, Gaunt T, Keith TP, Clough JB, Holgate ST, Holloway JS: Polymorphisms in the IL-4 and IL-4 receptor alpha chain genes confer susceptibility to asthma and atopy in a Caucasian population. Clin Exp Allergy 33:1111, 2003. Cui T, Wu J, Pan S, Xie J: Polymorphisms in the IL-4 and IL-4R (alpha) genes and allergic asthma. Clin Chem Lab Med 41:888, 2003.