The Journal of Allergy and Clinical Immunology
Volume 122, Issue 4 , Pages 726-733.e3, October 2008

Cytokine response after severe respiratory syncytial virus bronchiolitis in early life

  • Mario Castro, MD, MPH

      Affiliations

    • Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Washington University School of Medicine, St Louis, Mo
    • Corresponding Author InformationReprint requests: Mario Castro, MD, MPH, Washington University School of Medicine, Campus Box 8052, 660 S Euclid, St Louis, MO 63110-1093.
    • ,
  • Toni Schweiger, RN

      Affiliations

    • Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Washington University School of Medicine, St Louis, Mo
    • ,
  • Huiquing Yin-DeClue, PhD

      Affiliations

    • Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Washington University School of Medicine, St Louis, Mo
    • ,
  • Thiruvamoor P. Ramkumar, PhD

      Affiliations

    • Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Washington University School of Medicine, St Louis, Mo
    • ,
  • Chandrika Christie, BA

      Affiliations

    • Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Washington University School of Medicine, St Louis, Mo
    • ,
  • Jie Zheng

      Affiliations

    • Department of Biostatistics, Washington University School of Medicine, St Louis, Mo
    • ,
  • Rebecca Cohen, BA

      Affiliations

    • Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Washington University School of Medicine, St Louis, Mo
    • ,
  • Kenneth B. Schechtman, PhD

      Affiliations

    • Department of Biostatistics, Washington University School of Medicine, St Louis, Mo
    • ,
  • Robert Strunk, MD

      Affiliations

    • Department of Pediatrics, Washington University School of Medicine, St Louis, Mo
    • ,
  • Leonard B. Bacharier, MD

      Affiliations

    • Department of Pediatrics, Washington University School of Medicine, St Louis, Mo

Received 14 February 2008; received in revised form 8 July 2008; accepted 11 July 2008. published online 29 August 2008.

Article Outline

Background

Immune response after viral infection usually involves TH1-mediated response; however, severe respiratory syncytial virus (RSV) infection appears to be associated with the development of asthma, a TH2-predominant phenotype.

Objective

To understand the early and subsequent immunologic response to a serious RSV infection in children over time.

Methods

A total of 206 previously healthy infants hospitalized with severe RSV bronchiolitis were enrolled in a prospective cohort called the RSV Bronchiolitis in Early Life study. Peripheral blood T cells were obtained immediately after RSV infection and at 2, 4, and 6 years of age, stimulated with phorbol 12-myristate 13-acetate and ionomycin, and analyzed for IL-2, IL-4, IL-13, and IFN-γ by flow cytometry and real-time PCR.

Results

Of the children, 48% (n = 97) developed asthma (physician-diagnosed), and 48% (n = 97) had eczema by age 6 years; 32% (n = 48 of 150) developed allergic sensitization by 3 years of age. Children with asthma had lower IL-13 expression at 6 years of age than those without (P = .001). IFN-γ, IL-2, and IL-4 levels did not differ by asthma or eczema status during follow-up (all P > .05). Allergic sensitization was not associated with differences in cytokine levels during follow-up (all P > .05).

Conclusion

Severe RSV infection early in life is associated with a high incidence of asthma and eczema. Contrary to expectations, subsequent immunologic development in those who developed asthma, eczema, or allergic sensitization was not associated with a TH2 phenotype in the peripheral blood.

Key words: RSV bronchiolitis, asthma, eczema, allergic sensitization, cytokines

Abbreviations used: FACS, Fluorescence-activated cell sorting, RBEL, RSV Bronchiolitis in Early Life study, RSV, Respiratory syncytial virus

 

Respiratory syncytial virus (RSV) is a common early life pathogen, infecting 95% of children by age 2 years.1 However, 1% to 3% of children develop severe bronchiolitis requiring hospitalization.2 RSV bronchiolitis is the leading cause of hospitalizations in infants ≤1 year of age and costs $750 million annually.3, 4 Although infants who are premature, have bronchopulmonary dysplasia, or have congenital heart disease are at increased risk of developing severe RSV infection, the majority (53%) of hospitalizations caused by RSV bronchiolitis occur in previously healthy full-term infants.2 Ongoing respiratory symptoms are common after severe RSV bronchiolitis, with approximately 40% of children subsequently developing asthma in a previous case-controlled study.5, 6, 7

Asthma is a chronic respiratory disorder characterized by airway inflammation, hyperreactivity, and remodeling of the airways. Asthma affects 22 million people in the United States and accounts for more than $12 billion in direct and indirect health care costs per annum.8 The etiology of asthma is complex, involving genetics, environmental exposures (including infections), and the host's immunologic response.

Immune development early in life is a multifaceted interaction of genetic, molecular, and cellular components, regulatory elements, and complex pathways that is variable by type of infection and age. In the context of asthma, there appear to be underlying abnormalities in the general immune response and local immune dysregulation in the mucosal surfaces.9 One of the specific characteristics of asthma is an imbalance in TH1-predominant and TH2-predominant immune responses. In the airways of people with asthma, evidence suggests that there in an increase in IFN-γ and a decrease in IL-4.10 Furthermore, elevated IL-4 and IFN-γ in cord blood was associated with a physician's diagnosis of asthma at age 6 years.11 Therefore, cytokine measures in early life may be helpful in identifying those individuals at increased risk of developing asthma.

To evaluate the link between severe RSV infection and subsequent asthma development, we enrolled 206 infants hospitalized with their first episode of RSV bronchiolitis into the RSV Bronchiolitis in Early Life (RBEL) cohort study and followed them prospectively through 6 years of age. In the current study, we investigated the relationship between cytokine response at initial RSV infection and over time with the development of asthma, allergic sensitization, and eczema. We hypothesized that severe RSV infection resulting in bronchiolitis would stimulate a persistent TH2-predominant response profile with elevated IL-4 and IL-13 production at 6 years of age in children who had developed asthma.

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Methods 

Participants 

Details of the inclusion/exclusion criteria and baseline characteristics of the RBEL cohort have been published previously.12 At Saint Louis Children's Hospital, from 1998 to 2001, a total of 1222 infants with a nasopharyngeal swab positive for RSV were identified. Infants were included if they were ≤12 months old, had a first episode of wheezing (documented by a physician), were otherwise healthy, had bronchiolitis severe enough to require emergency department care or hospitalization, and had a confirmatory nasopharyngeal swab positive for RSV. There were 322 infants eligible to participate; of those, 206 infants were enrolled in the RBEL cohort and followed prospectively. The parents or legal guardians of the children were contacted at 3-month intervals by telephone to assess asthma, eczema, and wheezing episodes in the child. Study visits were conducted annually. The data were entered into a FileMaker Pro 6 database (Santa Clara, Calif). Informed consent was obtained from the parent/legal guardian, and the Washington University Medical Center Institutional Review Board approved the study.

Flow cytometry 

Blood samples were obtained at entry (n = 131; 140 ± 101 days old) and at 2 (n = 113), 4 (n = 107), and 6 (n = 101) years of age in a subset of subjects enrolled in the RBEL cohort. PBMCs were isolated from the whole blood by using Ficoll-Hypaque (GE Healthcare, Piscataway, NJ) and placed at 1 × 106 cells/mL/10 mm well in RPMI 1640 medium supplemented with 7.5% FBS, L-glutamine, nonessential amino acids, sodium pyruvate, 2-mercaptoethanol, and penicillin/streptomycin. The PBMCs were then incubated at 37°C in the presence of 10 μL/mL brefeldin A in the presence and absence of phorbol 12-myristate 13-acetate (25 ng/mL) and ionomycin (1 μL/mL) for 4 hours at 37°C. Cells were then labeled with fluorescein isothiocyanate–labeled or phycoerythrin-labeled mAbs to the T-cell surface markers CD3, CD4, and CD8, then permeabilized and incubated with mAbs directed against IL-2, IL-4, IL-13, and IFN-γ (all from Becton Dickinson Biosciences, San Jose, Calif). The samples were then analyzed on a dual channel FACScalibur flow cytometer (Becton Dickinson Biosciences) to determine percent positive cells in each sample based on a minimum of 10,000 events in live cell gate/condition and corrected for background detected with isotype-matched control for primary mAbs.

Real-time PCR 

RNA was extracted from PBMC pellets after stimulation with phorbol 12-myristate 13-acetate and ionomycin by using the RNEasy kit (Qiagen Inc, Valencia, Calif) according to the manufacturer's recommendations. cDNA was then synthesized from the extracted RNA by using the TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, Calif) containing Oligod(T)16, Multiscribe Reverse Transcriptase, and AmplTaq Gold DNA polymerase. Real-time PCR was later performed on the cDNA to assay for the expression levels of IL-2, IL-4, IL-13, IFN-γ, and glyceraldehyde-3-phosphate dehydrogenase. Individual reactions were set up in universal PCR master mix with an optimized forward primer, reverse primer, and 6FAM linked probe combination for each gene that was assayed (Applied Biosystems). The reactions were then run on a GeneAmp 5700 sequence detection system from Applied Biosystems. The output RNA levels from the real-time PCR reaction were then standardized to unit levels of GAPDH expression.

Allergic sensitization 

Allergy skin testing was conducted at the 3-year follow-up in 150 children from the RBEL cohort. Children underwent skin prick testing using the MultiTest II (Lincoln Laboratories, Lincoln, Ill) with a panel of locally prevalent aeroallergens, including dust mite mix, cat (standardized), dog (mixed breed), German cockroach, Penicillium species mix, Aspergillus species mix, timothy grass, short ragweed (standardized), eastern tree mix, and food allergens, including egg, milk, and peanut (Greer Labs, Lenoir, NC) as described by the Childhood Asthma Management Program.13 A wheal of ≥3 mm greater diameter than the negative saline control was considered positive. Children with a history of allergies to egg, milk, or peanut (n = 4) were tested by using an in vitro assay for allergen-specific IgE (ImmunoCAP), with an allergen specific IgE level of >0.35 kU/L considered indicative of sensitization. Children with sensitivity to 1 or more allergens by either skin test or in vitro testing were considered to have allergic sensitization.

Asthma 

Asthma was defined by having a diagnosis of asthma from a physician by parental report confirmed by review of the medical records. An affirmative response at any point during the 6 years of follow-up was carried forward. Participants who never responded in the affirmative were considered not to have asthma.

Eczema 

At enrollment and at each subsequent contact during the study, parents or legal guardians of the participant were asked whether the child had been diagnosed with eczema by a physician. A participant was considered to have eczema if (1) the participant was diagnosed before entry into the cohort, or (2) the participant was diagnosed at any point during the 6 years of follow-up.

Statistical methods 

Cytokine levels obtained by flow cytometry (FACS) are presented as the mean number of positive cells, and the levels obtained by real-time PCR are presented as pg/GAPDH. The cytokine levels for both FACS and real-time PCR were not normally distributed; therefore, they were log-transformed for analysis. The Spearman coefficient was used to assess the correlation between cytokine levels at entry and 2 years, 4 years, and 6 years after entry. Differences in cytokine levels over time were assessed by using a mixed-model repeated-measures ANOVA. We have used the mixed-model approach because it allows for the presence of missing data and different patterns of correlation across time points. For comparisons between asthma and nonasthma, eczema and noneczema, allergic sensitization and no allergic sensitization, and age at initial infection (≤6 mo/>6 mo), the Wilcoxon test or χ2 was used where appropriate. A P value <.05 was considered significant. All analyses were conducted in SAS version 9.1 (Cary, NC).

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Results 

Participant characteristics 

At the time of initial RSV bronchiolitis, 59 (29%) participants were 3 months of age or younger, and 96 (47%) were 6 months of age or younger, with a mean age at initial infection of 4.3 ± 3.3 months. Nonwhite ethnic groups represented 48% of the cohort, and 58% were boys (Table I). Forty-five percent of the cohort had ≥1 first-degree relative with allergies, and 43% had ≥1 first-degree relative with a history of asthma. Twenty-seven percent of the cohort had ≥1 first-degree relative with a history of eczema, and 21% had ≥1 first-degree relative with a history of hay fever.

Table I. Asthma, eczema, and allergy sensitization in children with severe RSV bronchiolitis in early life
AsthmaEczemaAllergy sensitization
Overall (n = 206)Yes (n = 97)No (n = 104)P valueYes (n = 97)No (n = 104)P valueYes (n = 48)No (n = 102)P value
Race, n (%)
African American9050 (52)37 (36).0642 (43)45 (43).3225 (52)38 (37).23
White10843 (44)63 (61) 49 (51)57 (55) 21 (44)60 (59)
Other84 (4)4 (4) 6 (6)2 (2) 2 (4)4 (4)
Male, n (%)120 (58)51 (53)66 (64).1257 (59)60 (58).8831 (65)56 (55).22
Log IgE level, mean (SD)2.52 (0.90)2.53 (0.96)2.51 (0.86).882.61 (0.96)2.44 (0.85).212.61 (1.08)2.51 (0.8).58
Eosinophils, n (SD)210.7 (237.9)199.1 (255.5)220.3 (223.2).56227.6 (274.3)196.6 (203.3).39169.5 (183.6)223.4 (251.6).21
Lowest O2 saturation91.7 (7.3)92.4 (6.0)91.0 (8.2).1791.5 (8.2)91.9 (6.1).7093.1 (6.9)91.3 (6.5).13

Sample size for each category: log IgE, overall n = 186, asthma n = 88, no asthma n = 95, eczema n = 89, no eczema n = 94, allergy sensitization n = 45, nonallergy sensitization n = 96; eosinophils, overall n = 174, asthma n = 79, no asthma n = 95, eczema n = 79, no eczema n = 95, allergy sensitization n = 41, nonallergy sensitization n = 133; lowest O2 saturation, overall n = 191, asthma n = 92, no asthma n = 94, eczema n = 91, no eczema n = 95, allergy sensitization n = 44, no allergic sensitization n = 92.

Forty-eight percent (n = 97) of the cohort had physician-diagnosed asthma by 6 years of age, and 48% (n = 97) had eczema at entry or diagnosed subsequently. There were no statistically significant difference in demographics, IgE levels, and peripheral blood eosinophil counts at initial infection, or severity of bronchiolitis (lowest O2 saturation or length of stay) between those with physician-diagnosed asthma and those participants who had not received a physician diagnosis of asthma by the age of 6 years (all P values >.05), although African-American subjects had a borderline higher likelihood of physician-diagnosed asthma (52% vs 44% among white subjects; P = .06). Participants who had eczema at entry (n = 25) or developed eczema during follow-up (n = 72) were similar to those who did not have eczema at either time in terms of baseline demographic characteristics, immunity, and disease severity (all P > .05; Table I).

Thirty-two percent (n = 48) of those tested for allergic sensitization at 3 years of age (n = 150) were sensitized to at least 1 allergen, with 31% demonstrating sensitization to aeroallergens and 9% to foods. There was no statistical difference in demographics, family history, or asthma outcomes between those who underwent allergy skin testing and those participants who did not at age 3 years, except that children with a mother who had hay fever were more likely to have undergone testing (P = .03).

Cytokine changes over time 

The age of child during the initial infection was not associated with either a predominant TH1 or TH2 cytokine response profile at entry (Table II). As the RBEL cohort aged to 6 years old, there was an overall increase in production of TH2 cytokines over time, as well as a decrease in TH1 cytokines (Table III).

Table II. Age at initial infection, cytokine response, and outcomes∗
≤6 mo>6 moP value
Cytokine levels at entry (by flow cytometry); mean % positive cells (SD)∗n = 96n = 35
IL-213.0 (14.4)9.7 (9.2).13
IL-41.5 (5.1)1.0 (1.5).40
IL-130.8 (1.5)0.8 (0.7).89
IFN-γ2.9 (6.8)2.7 (3.3).83

3-y outcomen = 69n = 28
≥1 positive allergy skin test; n (% positive)27 (39)6 (21).10

6-y outcome, n (%)n = 94n = 35
Doctor-diagnosed asthma54 (58)12 (34).02
Eczema43 (46)22 (63).08

∗N = 131 subjects in which peripheral blood was available for cytokine measurements.

Table III. Cytokine expression (by flow cytometry) by age
CytokineEntry (N = 131)2 y (N = 113)4 y (N = 107)6 y (N = 101)Overall∗P valueEntry/2 y vs 6 y P value
IL-212.1 (13.2)6.8 (7.7)3.6 (5.3)0.8 (2.2)<.0001<.001
IL-41.4 (4.5)0.8 (1.1)6.0 (6.7)12.6 (7.4)<.0001<.001
IL-130.8 (1.3)1.1 (1.5)4.3 (5.3)4.1 (3.0)<.0001<.001
IFN-γ2.9 (6.0)9.4 (10.9)5.2 (6.2)1.4 (2.8)<.0001<.001

Data expressed in mean % positive cells.

∗ANOVA P value for overall comparison and between the initial infection and 6 y.

For the TH1 cytokines, IL-2 levels were highest immediately after infection and decreased until 6 years of age (P < .001). IFN-γ levels for the entire cohort increased from immediately after infection to 2 years, then decreased from 2 years to 6 years of age. There is a significant difference between the IFN-γ levels after infection and 2 years of age (P < .001) and 2 years compared with 6 years of age (P < .001). Overall, there was a decrease in the IFN-γ levels from immediately after infection to 6 years of age (P < .001; Table III).

For the TH2 cytokines, the IL-4 levels increased over time (P < .001), as did IL-13 levels (P < .001; Table III).

Flow cytometry compared with real-time PCR 

Fluorescence-activated cell sorting was performed immediately after infection and at 2, 4, and 6 years of age. The real-time PCR was performed starting at 2, 4, and 6 years of age because this technology became available after the start of the study. The IL-2 levels obtained by FACS versus real-time PCR were correlated at 2 years (r = 0.50; P = .05) and at 4 years of age (r = 0.52; P < .0001) but not at 6 years of age (r = 0.01; P = .95). The IFN-γ levels measured by FACS and real-time PCR were significantly correlated for all time points: 2 years (r = 0.52; P = .04), 4 years (r = 0.49; P < .0001), and 6 years of age (r = 0.38; P = .002). The IL-4 levels by FACS and real-time PCR were not correlated at 2 years of age (r = 0.09; P = .73) but were correlated at 4 years (r = 0.23; P = .02) and 6 years of age (r = 0.35; P = .004). IL-13 measurements by FACS and real-time PCR did not correlate at 2 years (r = 0.12; P = .66), 4 years (r = 0.04; P = .71), or 6 years of age (r = 0.22; P = .08). See Fig E1, Fig E2 through E3 in the Online Repository at www.jacionline.org for Bland Altman plots correlating cytokine expression levels by flow cytometry and real-time PCR at 2, 4, and 6 years of age.

TH1 and TH2 cytokine profiles and immune development 

The TH1 cytokines (IL-2 and IFN-γ) were correlated with each other, and the TH2 cytokines (IL-4 and IL-13) were correlated with each other at the initial infection and 2 years, 4 years, and 6 years of age by FACS (all P < .05; Table IV). During initial infection, IL-2 was weakly correlated with IL-13 (r = 0.24; P = .0005), and IFN-γ was weakly correlated with both IL-4 (r = 0.27; P = .002) and IL-13 (r = 0.26; P = .003); however, by 6 years of age, these relationships were no longer significant. This suggests that the maturations of TH1 and TH2 immunity are parallel and independent processes.

Table IV. Spearman correlation of mean number of cytokine-positive cells during follow-up
IL-4 CoefficientP valueIL-13 CoefficientP valueIFN-γ CoefficientP value
IL-2
Entry0.158.0700.244.00050.589<.0001
2 y0.326.00040.493<.00010.808<.0001
4 y0.070.4740.329.00050.800<.0001
6 y−0.193.0530.094.3500.334.0007
IL-4
Entry 0.349<.00010.269.002
2 y 0.394<.00010.267.004
4 y 0.547<.00010.057.557
6 y 0.472<.0001−0.202.043
IL-13
Entry 0.262.003
2 y 0.450<.0001
4 y 0.265.006
6 y −0.179.073

Cytokines and the development of asthma by 6 years of age 

Younger age during initial RSV infection (<6 mo) was associated with a higher prevalence of asthma at 6 years of age: 58% versus 34% in older children at entry (≥6 mo), respectively (P = .02). At the initial infection, TH1 or TH2 cytokine levels were not significantly different in children diagnosed with asthma at 6 years of age compared with those who were not diagnosed with asthma. For IL-2, IL-4, and IFN-γ, there were no differences in cytokine levels between those with and without physician-diagnosed asthma at any point during follow-up (all P > .05; Fig 1). At 6 years of age, there was a significant difference in IL-13 levels in children diagnosed with asthma and those who were not (3.2 ± 2.7 vs 5.0 ±3.1%; P = .001). The production of TH2 cytokines relative to TH1 cytokines, represented by the ratio of IL-4:IFN-γ, was not significantly different in those who developed asthma and those who did not (asthma vs nonasthma, 3.5 ± 10.2 vs 1.4 ± 2.8%; P = .14).

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  • Fig 1. 

    PBMC cytokine expression levels in those with and without asthma. Quantitative analysis of PBMCs by FACS for IL-2, IL-4, IL-13, and IFN-γ in those with physician-diagnosed asthma (gray bars) or not (white bars) in the RBEL cohort from initial infection with RSV (entry) to 2, 4, and 6 years of age. Means ± SEs. ∗P = .001.

Cytokines and the development of eczema by 6 years of age 

There were no significant differences in IFN-γ, IL-2, IL-4, and IL-13 cytokine levels at initial infection or at subsequent follow-up by eczema diagnosis status (all P > .05; Fig 2). Initially, the TH1 immune response in those who developed eczema lagged behind those who did not develop eczema, but as the immune response matured, the difference diminished with age.

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  • Fig 2. 

    PBMC cytokine expression levels in those with and without eczema. Quantitative analysis of PBMCs by FACS for IL-2, IL-4, IL-13, and IFN-γ in those with eczema (gray bars) or not (white bars) in the RBEL cohort from initial infection with RSV (entry) to 2, 4, and 6 years of age. Means ± SEs.

Cytokines and the development of allergy sensitization at 3 years of age 

Allergic sensitization, which was assessed at 3 years of age, was not associated with differences in cytokine levels at initial infection or 2 years after infection (all P > .05; Fig 3). After RSV bronchiolitis, cytokine profiles did not differ on the basis of allergic sensitization status.

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  • Fig 3. 

    PBMC cytokine expression levels in those with and without allergic sensitization. Quantitative analysis of PBMCs by FACS for IL-2, IL-4, IL-13, and IFN-γ in those with eczema (gray bars) or not (white bars) in the RBEL cohort from initial infection with RSV (entry) to 2, 4, and 6 years of age. Means ± SEs.

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Discussion 

The RBEL cohort offers a unique opportunity to follow the immune development, specifically the changes in the TH1 and TH2 cytokine production, during the first 6 years of life after severe RSV bronchiolitis, and to investigate the subsequent development of asthma, allergic sensitization, and eczema. In this study we found that TH1 cytokines in PBMCs tend to decrease and TH2 cytokines tend to increase over time after initial severe RSV bronchiolitis. Interestingly, the level of these immunologic parameters in PBMCs does not appear related to the development of asthma, allergic sensitization, or eczema. Younger age at initial infection was not associated with different cytokine levels immediately after infection, subsequent allergic sensitization, or the development of eczema, although it was associated with development of asthma by 6 years of age. TH1 or TH2 cytokine response during initial infection was not associated with the subsequent development of asthma, eczema, or allergic sensitization. These findings advance our knowledge of the complex developmental immune response in children after a severe infection early in life and suggest that newer immunologic approaches, including studying the response at the local end-organ level and in different immune cell populations, are needed.

Immune response to a viral pathogen such as RSV involves many different cells types (eg, lymphocytes, natural killer cells), chemotaxins, and signaling pathways. For the purposes of this investigation, we focused on cytokine levels in the peripheral blood during the initial infection and as the child developed over the subsequent 6 years. Cytokine response is commonly divided into 2 categories—TH1-predominant and TH2-predominant—depending on the cytokine profile produced; however, other subgroups have been identified. The TH1 cytokine profile, which includes IL-2 and IFN-γ, is associated with successful clearing of pathogens. The TH2 response, characterized by the production of IL-4 and IL-13, is associated with atopic diseases including asthma. These 2 cytokine profiles appear to be counterregulatory or cross-regulated,14 and deviation from TH2 or toward TH1 is associated with a more severe disease state in several experimental disease models.15 However, this dichotomous view of TH1 versus TH2 does not hold as well in human beings as in mice.16, 17

Recent evidence also suggests that the immune response during RSV infection may be influenced by age. Ichinohe et al18 demonstrated that the capacity to produce IL-4 and IFN-γ in response to RSV infection increases with age. In our data, cytokine responses to initial RSV infection did not differ by age during infection. The differences in findings could be related to cellular location of cytokine production and age of the study population. In another study, Chung et al19 found that younger children with RSV infection (<6 months old) had diminished IFN-γ levels. In our study, we found that children infected at younger than 6 months of age were at increased risk of developing asthma at 6 years of age; however, they did not have significantly lower levels of INF-γ compared with the older children (Table II). These differences may be explained by the larger sample size in our cohort or differences in technique of measuring cytokine levels in peripheral blood.

Intrauterine and early postnatal TH1 response in healthy nonatopic children is immature, and the hormones present in the placenta promote TH2 cytokine responses.20 Studies have demonstrated the ability to produce IL-4 and IFN-γ cytokines increases with age in healthy infants.18, 21, 22 The evidence for IL-2 in healthy infants is less clear. Härtel et al21 showed by both mRNA transcripts and flow cytometry in the PBMCs that IL-2 increased with age, but in other studies, IL-2 levels decreased with age.23, 24

Similarly, after RSV infection, there appears to be a decrease in TH1 cytokines (IL-2 and IFN-γ) and an increase in TH2 cytokines (IL-4 and IL-13). However, this increase in TH2 cytokine levels may not be unique to RSV bronchiolitis and may be related to the maturation of the immune system.25, 26 Kristjansson et al27 found that younger infants with RSV had increased production of IL-4 compared with healthy age-matched controls, but that this trend was also seen after influenza or parainfluenza infection. Furthermore, in early life, TH1 and TH2 cytokine levels appear to be correlated. One study using cord blood found that IL-4 was correlated with both IL-13 and IFN-γ.11 Our data support this observation based on the finding that PBMC IL-4 expression was correlated with IL-13 and IFN-γ at the time of initial infection with RSV (Table IV), but over time this relationship weakens. It appears that the adaptive immune response is age-dependent, but the TH2 bias, which occurs early in young children, is not unique to RSV infection.

In the current study, we hypothesized that severe RSV infection resulting in bronchiolitis would stimulate a persistent TH2 response profile with elevated IL-4 and IL-13 production at 6 years of age in those children who had developed asthma. Attenuated production of IFN-γ has also been associated with increased risk for allergic sensitization14, 22, 28, 29 or asthma30 early in life, although this relationship appears not to hold true later in childhood. In contrast, detectable levels of cord blood IFN-γ and IL-4 were protective against developing asthma at 6 years.11 In our study, we found no significant difference in TH1 or TH2 cytokine production at the initial RSV infection in those who developed asthma or allergic sensitization by 6 years of age compared with those who did not. Furthermore, the production of TH2 cytokines relative to TH1 cytokines, represented by the ratio of IL-4:IFN-γ, was not different in those who developed asthma compared with those who did not. In contrast with previous reports,14, 22, 28, 29 children in our study who had or developed eczema by 6 years of age had slightly lower IFN-γ levels during the initial infection than participants who did not develop eczema but this did not achieve significance. Lack of finding an association of peripheral blood cytokine levels with asthma, allergic sensitization, or eczema in our study may be related to different methodologies of measuring cytokine levels in PBMCs, the PBMCs not reflecting airway or lung tissue levels, inadequate power in our study to detect small differences, or it may simply reflect that expression of cytokine levels in the peripheral blood post-RSV bronchiolitis is not predictive of the subsequent development of asthma or allergic sensitization. Previous studies have found that flow cytometry and real-time PCR are adequate for detecting cytokines in stimulated blood with high intratest and intertest reliability,31, 32, 33, 34, 35 although others have used ELISA-based assays from cultured PBMCs. Similar to previous studies,36, 37 we found variable correlation between flow cytometry and real-time measurement of peripheral blood cytokine levels depending on the age at which the measurements were made. Other immunologic responses (eg, antigen-specific responses14) or subpopulations of immune cells (eg, dendritic cells38, 39, 40) may be important in determining the pathogenesis of asthma after RSV infection.

Ideally one would want to compare immune development in infants without RSV infection and/or RSV infection without severe bronchiolitis to those who have severe bronchiolitis with RSV. Because of the ubiquitous nature of RSV1 and difficulties in identifying nonsevere RSV cases in the general population at the time of infection, a proper control group could not be identified. Therefore, the results of this study are to be interpreted in the context of RSV with severe bronchiolitis.

Drawing on the strength of the prospective RBEL cohort, we were able to follow immune development in a large cohort of children during the first 6 years of life after severe RSV bronchiolitis. In this current study, we sought to investigate TH1 and TH2 immunity in early life, the change in immunity over time, and their role in the subsequent development of asthma, allergic sensitization, and eczema after severe RSV bronchiolitis. We found that TH1 cytokines tend to decrease over time after initial severe RSV bronchiolitis and TH2 cytokines tend to increase over time, but these patterns were unrelated to asthma and allergy outcomes. The magnitude of TH1 and TH2 cytokine responses during RSV infection was not associated with the subsequent development of asthma, eczema, or allergic sensitization. Given the observed high incidence of asthma after severe RSV bronchiolitis, there is significant need for new insights into the pathogenesis of this illness so that optimal preventative strategies can be developed.

Key message


•PBMC cytokine levels after severe RSV bronchiolitis are not predictive of asthma or allergic sensitization by 6 years of age.

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We thank Wynona Black, MPH, for her statistical assistance and contribution to this work; Lisa Robertson, RN, Lynette Tegtmeier, RN, Amy Rahm, RN, Michelle Jenkersen, RN, RRT, JoAnn Bonfiglio, RN, MSN, and Toni Schweiger, RN, for their assistance in recruiting children and data collection for the RBEL study; and, most importantly, the children and their families who graciously provided their time and effort to participate in the RBEL study.

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Fig E1. 

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  • Bland-Altman plots of PBMC cytokine expression levels measured by flow cytometry and real-time PCR in the RBEL cohort children at 2 years of age for IFN-γ (A), IL-2 (B), IL-4 (C), and IL-13 (D).

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Fig E2. 

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  • Bland-Altman plots of PBMC cytokine expression levels measured by flow cytometry and real-time PCR in the RBEL cohort children at 4 years of age for IFN-γ (A), IL-2 (B), IL-4 (C), and IL-13 (D).

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Fig E3. 

  • View full-size image.

  • Bland-Altman plots of PBMC cytokine expression levels measured by flow cytometry and real-time PCR in the RBEL cohort children at 6 years of age for IFN-γ (A), IL-2 (B), IL-4 (C), and IL-13 (D).

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 Supported by National Institutes of Health grant no. HL 61895.

 Disclosure of potential conflict of interest: The authors have declared that they have no conflict of interest.

PII: S0091-6749(08)01318-3

doi:10.1016/j.jaci.2008.07.010

The Journal of Allergy and Clinical Immunology
Volume 122, Issue 4 , Pages 726-733.e3, October 2008

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