Specific induction of interleukin-4-producing cells in response to in vitro allergen stimulation in atopic individuals

Background and Objective CD4⁺ T cells can be divided into two major subsets. T helper (TH)₁ and TH₂ cells. Interleukin-4 (IL-4) is produced by TH₂ cells and induces switching of immunoglobulin (Ig) M/lgG to IgE. Interferon-γ (IFNγ) produced by TH₁ cells counteracts the IgE-promoting effects of IL-4. In this study we wanted to investigate whether the number of IL4-producing cells could be a direct measurement of allergen exposure in vitro, and whether this was correlated to the elevated serum IgE-levels seen in atopic persons. Methods We compared the number of IL-4- and IFNγ-producing cells using an enzyme-linked immunospot assay (ELISPOT) in response to allergens from birch and cat in peripheral mononuclear cells from atopic and healthy individuals. Results In the two sensitized groups there was an increase in the number of lL-4producing cells in response to the specific allergen which was not seen in the healthy group (1/20000 cells and 1/200 000 cells, respectively, P < 0.001 for birch). In criss-cross experiments where birch-sensitized individuals were stimulated with cat allergen, no lL-4-producing cells were seen, indicating a high degree of specificity. In individual subjects, the elevated numbers of IL-4-producing cells were significantly correlated with their allergen-specific serum IgE levels. When allergen was combined with a suboptimal dose of PHA, there was a synergistic increase in the number of allergen-induced IL-4-producing cells (1/4000 cells) in the atopic donors, which was not seen with the number of IFNγ-producing cells. Conclusions Allergen-specific IL-4 producing cells in a peripheral blood mononuclear cell (PBMC) culture can be detected by ELISPOT and the response can synergistically be enhanced by suboptimal concentrations of PHA.     

Low numbers of interleukin-12-producing cord blood mononuclear cells and immunoglobulin E sensitization in early childhood

BACKGROUND: Successful pregnancies are associated with skewing towards a Th2 cytokine profile. Cytokine responses to allergens can be detected in cord blood mononuclear cells (CBMC), suggesting allergen priming already in utero. OBJECTIVE: To investigate the cytokine profile in CBMC after in vitro stimulation with allergens and to relate the responses to the outcome in terms of allergic disease at 2 years of age. METHODS: CBMC were isolated from 82 children. The responses to ovalbumin (OVA), birch, cat and phytohaemagglutinin (PHA) were investigated by the ELISpot technique. The numbers of IFN-gamma-, IL-4- and IL-12-producing CBMC were counted for each stimulation. The children were followed prospectively; skin prick test (SPT) and RAST towards food and inhalant allergens were assessed at 24 months of age. RESULTS: Sixteen (19.5%) children were classified as IgE sensitized (positive SPT; > or =3 mm and/or RAST; > or =0.35 kUA/L). The numbers of IL-12-producing CBMC after stimulation with birch, OVA and cat were lower among IgE-sensitized children, statistically significant for cat. IFN-gamma-producing cells, did not differ in numbers between the sensitized and non-sensitized children. Children who had atopic eczema/dermatitis syndrome (AEDS) during the observation (n=53) had significantly lower numbers of IFN-gamma-producing CBMC after stimulation with OVA and cat than their non-AEDS counterparts. CONCLUSIONS: Although the numbers of infants in our study are limited our data suggest that a low number of IL-12-producing CBMC is associated with IgE sensitization during early childhood and that a reduced number of IFN-gamma-producing CBMC promotes the development of AEDS during the first 2 years of life.     

IFN-γ but not IL-4 T cells of the asthmatic bronchial wall show increased incidence of apoptosis

BACKGROUND: Previous observations have established that IFN-gamma production is depressed in CD4+ T cells from atopic asthmatics compared with non-asthmatics. OBJECTIVE: The aim of this study was to determine if decreased IFN-gamma production could be due to a dissociation between levels of apoptosis within the T cell subsets of the asthmatic bronchial wall. METHODS: Twenty asthmatics (10 atopic and 10 non-atopic) and eight non-atopic non-asthmatics underwent bronchoscopy. Cryostat sections of these biopsies were investigated using immunohistological techniques to determine the relative number of CD4/FAS+ and CD4/Bcl-2+ cells. Detection of IFN-gamma+ and IL-4+ was combined with TUNEL staining to determine the proportions of the Th1 and Th2 cells undergoing apoptosis. RESULTS: Experiments revealed raised proportions of activated CD4+ T cells as assessed by expression of HLA-DR and CD25+ expression in the asthmatic samples. Expression of Bcl-2 by the CD4+ cell population was significantly reduced in the asthmatic compared with the control group (P = 0.002). There was no significant difference in the expression of CD4+ Fas-ligand or the number of CD4+ undergoing apoptosis in the asthmatic and non-asthmatic groups. However, the IFN-gamma+ (P = 0.04) but not IL-4+ T cells in the asthmatic biopsies had significantly higher proportions of apoptotic cells compared with the control group. CONCLUSION: The evidence supports the hypothesis that Th1/Th2 imbalance in asthmatic inflammation may be a result of premature apoptosis within the Th1 subset.     

Serum concentrations of interferon-γ and intercellular adhesion molecule-1 eight years after an early respiratory syncytial virus infection

BACKGROUND: Respiratory syncytial virus (RSV) infection may influence the development of recurrent wheezing and atopy, but the mechanisms are unclear. OBJECTIVE: The purpose was to evaluate serum concentrations of soluble intercellular adhesion molecule-1 (sICAM-1), CD14, IgE, IL-5 and IFN-gamma in children 6-10 years after an RSV infection and their correlation with subsequent asthma and atopy. METHODS: Fifty-one subjects admitted to hospital for RSV infection during the first year of life and controls matched for birth date and sex underwent clinical examinations including lung function, skin prick and blood tests. RESULTS: The RSV subjects had significantly higher serum concentrations of IFN-gamma and sICAM-1 than the controls (for IFN-gamma 224.9 pg/mL (standard deviation (SD) 271.3) vs. 187.1 pg/mL (372.9), difference 37.8 pg/mL, 95% confidence interval (CI) -90.3 to 166.0, P = 0.05; for sICAM-1 170.2 ng/mL (SD 63) vs. 147.8 ng/mL (SD 57), difference 22.4 ng/mL, 95% CI -1.4 to 46.1, P = 0.04). The RSV subjects with asthma had significantly higher concentrations of IFN-gamma than the controls with asthma, and the RSV subjects with wheezing during the previous 12 months had significantly higher concentrations of both IFN-gamma and sICAM-1 than the controls with wheezing. CONCLUSIONS: Children hospitalized for RSV infection in infancy still differ in IFN-gamma and sICAM-1 production 6-10 years after the infection. The data suggest that the pathomechanism of asthma and wheezing after an early RSV infection may be different from that of children without an early RSV infection.     

Developmental changes in interleukin-12-producing ability by monocytes and their relevance to allergic diseases

BACKGROUND: The T helper type-2 (Th2)-dominated situation can be observed in allergic diseases such as asthma or atopic dermatitis. A reduced ability to produce IL-12, which is a key cytokine for the induction of Th1 responses, has been proposed to lead to aberrant Th2 development in these disease conditions. OBJECTIVE: This study was intended to examine how IL-12-producing ability might associate with allergic diseases as a function of age. METHODS: IL-12 production by monocytes at various ages was assessed in patients with bronchial asthma and/or atopic dermatitis (n = 100) in comparison with non-allergic control subjects (n = 144). Whole blood cells were stimulated with lipopolysaccharide (LPS) after priming with IFN-gamma, then intracellular cytokine expression of IL-12 and IL-8 as a control cytokine of CD14-positive cells was assessed by flow cytometric analysis. RESULTS: In the control subjects, the ability of monocytes to produce IL-12 was negligible at birth and gradually increased with advancing age, whereas IL-8 production was intense throughout the human life. At more than 7 years of age, IL-12 production of patients with allergic diseases was significantly lower compared with that of control subjects. The unexpected finding was that infants and children below 6 years of age with allergic diseases tended to produce more IL-12 compared with age-matched controls. In this young group, it was noted that enhanced IL-12 production by monocytes was especially observed in allergic patients with specific IgE antibodies against some food allergens. Significant inverse relationships between serum IgE levels and IL-12-producing ability were found in the teenage and adult groups, but not in the younger children. CONCLUSION: IL-12 appeared to play different roles in the pathogenesis of allergic diseases between younger and older ages.     

病毒在哮喘发作的研究进展

哮喘的本质是气道的炎性反应,规律吸入糖皮质激素和β2受体激动剂可控制哮喘,但在此治疗基础上呼吸道病毒感染仍可导致哮喘的发作。固有及适应性免疫缺陷削弱抗病毒反应,而过敏性炎性反应有协同作用。随着对其机制的深入了解,为开辟新的防治提供新思路。     

Allergen challenge primes for IL-5 mRNA production and abrogates β-adrenergic function in peripheral blood T lymphocytes from asthmatics

BACKGROUND: In previous studies, we have found a dysfunctional adenylyl cyclase (AC) system in patients with asthma after allergen provocation, which resulted in a 40-50% decreased generation of intracellular cAMP. In addition, in activated T helper lymphocyte clones, it has been demonstrated that IFN-gamma (TH1-like cytokine) and IL-5 (TH2-like cytokine) are differentially regulated by the AC system. Therefore, we postulate that an increased IL-5/IFN-gamma ratio as observed in asthmatics might be due to a dysfunctional AC system. OBJECTIVE: To assess whether a dysfunctional AC system as observed in asthmatics after allergen provocation, is responsible for an increased IL-5/IFN-gamma cytokine ratio. METHODS: Peripheral blood T lymphocytes of seven asthma patients were stimulated with anti-CD3 plus anti-CD28 MoAbs in the absence and presence of isoproterenol (ISO) and prostaglandin E2 (PGE2) to activate the AC system. Before, 3 h and 24 h after allergen provocation, IFN-gamma and IL-5 mRNAs were detected by semiquantitative RT-PCR. RESULTS: Before allergen provocation, ISO (10-5 mol/L) significantly downregulated IFN-gamma mRNA (P < 0.03, n = 6), and showed a trend to upregulate IL-5 mRNA (P = 0.138, n = 5). Three and 24 h after allergen provocation, ISO was not longer able to modulate IFN-gamma and IL-5. In contrast with the observations with ISO, PGE2 still dose-dependently inhibited IFN-gamma mRNA, both before, 3 h and 24 h after allergen provocation (n = 7). IL-5 mRNA, but not IFN-gamma mRNA, was significantly upregulated in anti-CD3 plus anti-CD28-activated T cells (P < 0.05, n = 5) 24 h after allergen provocation, compared with before allergen provocation. CONCLUSION: Twenty-four hours after allergen provocation, a significant reduction of beta-adrenergic control on IFN-gamma and IL-5 mRNA expression was observed in peripheral blood T lymphocytes, which coincides with a selective priming of IL-5 mRNA production.     

Increased number of T cells committed to IL-5 production after respiratory syncytial virus (RSV) infection of human mononuclear cells in vitro

We examined changes in the cytokine profile of T cells induced by in vitro infection with RSV. Isolated mononuclear cells from 27 healthy adults and six infants were infected with RSV at a concentration of 3 MOI (multiplicity of infection). After 48 h cells were restimulated with phorbol ester and ionomycin in the presence of monensin for 5 h. The intracellular expression of viral antigen, the cytokines IL-2, IL-4, IL-5, interferon-gamma (IFN-γ), and the expression of surface markers were assessed by immunofluorescent staining and flow cytometry. There was a significant (P < 0.001) rise of IL-5 expression in RSV-infected cultures in comparison with uninfected cultures from the same individuals, whereas there were no changes in the expression of the other lymphokines. The increase in IL-5 generation depended on viable infectious RSV rather than inactivated virus. RSV infection as well as IL-5 production in T cells were confined to the CD8 subpopulation. However, there was no simultaneous expression of RSV antigen and IL-5. Purified T cells did not show any increase in IL-5 generation. However, when the rate of RSV infection was enhanced in monocytes by means of a specific monoclonal antibody, co-cultured T cells displayed an increase of IL-5 production compared with samples with ordinary low rate RSV infection. It is therefore likely that the increased commitment of lymphocytes to produce IL-5 after RSV infection in vitro is mediated by monocytes or other antigen-presenting cells.     

Association of an early respiratory syncytial virus infection and atopic allergy

BACKGROUND: Respiratory syncytial virus (RSV) causes postbronchiolitic wheezing but its role in allergic sensitization is controversial. The purpose of the study was to examine the effect of an early RSV infection on allergic sensitization. METHODS: Seventy-six subjects were examined 6-10 years after hospitalization for RSV infection during the first year of life. Fifty-one subjects (68%) attended clinical studies and 25 filled in a questionnaire. The study protocol included lung function, skin-prick and blood tests. The controls were matched for birth date and sex. RESULTS: Eight per cent of the subjects and 37% of the controls had at least one positive skin-prick test (SPT) (difference -35%, 95% CI -50 to -19%, P < 0.0001). Allergic rhinitis, atopic dermatitis and asthma occurred as often in both groups, but asthma had been diagnosed significantly earlier in the subjects than in the controls [mean age 3.0 years (SD 2.6) and 5.6 years (SD 3.0), difference 2.6 years, 95% CI 0.57-4.65, P = 0.014]. In a logistic regression analysis, RSV infection was associated with negative SPTs. CONCLUSIONS: An early RSV infection results in reduction of SPT positivity but not of occurrence of atopic diseases. This finding might explain why there is less atopic sensitization in countries with a greater probability of acquiring RSV infection at an early age.     

Specific immunotherapy prevents increased levels of allergen-specific IL-4- and IL-13-producing cells during pollen season

BACKGROUND: Specific allergen immunotherapy (SIT) is effective for treatment of IgE-mediated diseases: however, the mechanisms of action still remain unclear. Earlier, we showed that IL-4 and IL-13 are produced in response to specific allergens. The aim of this study was to investigate whether these cytokine responses were affected by allergen SIT, and, furthermore, to evaluate the effect of SIT on allergen-specific IgE and IgG4 levels. METHODS: Blood samples from pollen-sensitized individuals were collected before the pollen season (before treatment) and during the pollen season (after SIT or placebo treatment). Peripheral blood mononuclear cells were activated in vitro with allergens and the numbers of IL-4-, IL-13-, IL-10-, and IFN-gamma-producing cells were determined by ELISPOT. Serum levels of allergen-specific IgE and IgG4 were measured by RAST and ELISA, respectively. RESULTS: The numbers of IL-4- and IL-13-producing cells were shown to be increased in the placebo group during the pollen season, an increment which was absent in patients receiving allergen SIT. We found an increase in allergen-specific IgG4 in the SIT-treated individuals, but not in the placebo group. Both groups displayed elevated specific IgE levels during the pollen season. CONCLUSIONS: Taken together, our data show a downregulation of IL-4- and IL-13-producing cells in peripheral blood after SIT, suggesting induction of nonresponsiveness/tolerance or a redistribution of these cells. Furthermore, we demonstrate that SIT acts on antibody production by increasing the specific IgG4 levels.     

Allergen-induced interleukin-9 production in vitro: correlation with atopy in human adults and comparison with interleukin-5 and interleukin-13

BACKGROUND: The contribution of IL-9 to human atopy is supported by genetic studies. However, IL-9 production in response to allergen in vitro has been reported only in children. OBJECTIVE: Study IL-9 induction by allergen in adults, compare it with IL-5 and IL-13 and evaluate its association with atopy. METHODS: Peripheral blood mononuclear cell (PBMC) from control adults and from atopic patients were cultured with various allergens or phytohaemagglutinin (PHA) and secreted IL-5, IL-9 and IL-13 were measured by ELISA. RESULTS: IL-9 was produced in response to Dermatophagoides pteronyssinus (Der p) by PBMC from Der p-hypersensitive adults at levels equivalent to those induced by PHA but with slower kinetics. The induction of IL-9 was allergen specific, reflecting donor RAST profile. In Der p-triggered reactions of non-atopic and atopic subjects, IL-9 showed the highest selectivity for atopics, IL-5 and IL-13 being produced more frequently in non-atopic donors. Significant correlations with specific IgE titres were found for IL-9 with all allergens tested (Der p and two peptides of Bet v 1 birch allergen). For IL-5 and IL-13, they were in the same range for Der p but more variable for birch allergens. Patterns of cytokine production by individual patients in response to allergen reflected these differences: for Der p, IL-5, IL-9 and IL-13 productions were strongly correlated but for birch IL-5 differed from the latter two. The in vitro production of IL-9 reflected clinical hypersensitivity profiles and was higher in individuals with asthma than in those with disease limited to rhinitis and/or conjunctivitis. CONCLUSIONS: Allergen-triggered IL-9 production in vitro is an excellent marker for atopy in adults given its virtual absence in allergen-stimulated PBMC from non-atopic individuals and its correlation with allergen-specific IgE and asthma.     

Gene–gene interaction between interleukin-4 and interleukin-4 receptor α in Korean children with asthma

BACKGROUND: Interleukin-4 receptor alpha (IL-4Ralpha), which binds IL-4 and IL-13, is involved in signal transduction of those cytokines that lead to IgE production, and is also a key functional component of the Th2 lymphocyte phenotype. OBJECTIVE: To determine whether IL-4 and IL-4Ralpha polymorphisms are associated with susceptibility to asthma and whether there are gene-gene interactions between IL-4 and IL-4Ralpha polymorphisms. METHODS: We genotyped three groups of Korean children, consisting of 196 atopic asthmatics, 60 non-atopic asthmatics, and 100 healthy children, for an IL-4 promoter polymorphism (C-590T) and three IL-4Ralpha polymorphisms (Ile50Val, Pro478Ser, and Arg551Gln) using PCR-RFLP (restriction fragment length polymorphism) assays. RESULTS: The allele frequencies of the IL-4 (C/T) polymorphism and the Ile50Val and Pro478Ser polymorphisms of IL-4Ralpha did not differ statistically among the three groups of children. For the Arg551Gln polymorphism, the combined genotype frequency of the Arg/Gln heterozygote and the Arg/Arg homozygote was significantly higher in atopic asthmatics (27.6%) than in healthy children (16.0%) (odds ratio (OR) = 1.97, 95% CI (confidence interval) = 1.07-3.71). The eosinophil fraction (%) and bronchial responsiveness were higher in children with the Arg/Gln and Arg/Arg genotype than in those with the Gln/Gln genotype (P = 0.036 and 0.024, respectively). In asthmatic children, combinations of the IL-4 CT/TT genotype and the IL-4Ralpha Arg/Gln and Arg/Arg genotypes were associated with significantly increased risk for development of asthma (OR = 3.70, 95% CI = 1.07-12.78, P = 0.038). CONCLUSIONS: In Korean children, the IL-4Ralpha Arg551 allele may play a role in susceptibility to atopic asthma and correlate with markers of asthma pathogenesis, including increased eosinophil fraction and enhanced bronchial hyper-responsiveness. In addition, a significant gene-gene interaction between the IL-4-590C and the IL-4Ralpha Arg551 allele significantly increases an individual's susceptibility to asthma.     

Interleukin-4, interferon-gamma and interleukin-5 in peripheral blood of children with moderate atopic asthma

Background In asthmatic inflammation, TH2 cells play an important role. TH₂ cells specifically secrete cytokines like IL-4 and IL-5. IL-4 stimulates IgE production and IL-5 is involved in hemopoiesis, chemotaxis, priming and activation of eosinophils. IFNγ, produced by TH₁ cells, has an inhibitory action on IgE production. Objectives To investigate the TH₁/TH₂-cell pattern in the cytokine production of peripheral blood of asthmatic children. We determined IL-4, IFNγ and IL-5 in serum and in supematants of unstimulated and stimulated (24 h with Concanavaline A) cultures of peripheral blood mononuclear cells (PBMCs) in 22 children with moderate asthma (mean age 9.3 years) and in 17 healthy controls (mean age 10.3 years). All children visited the outpatient department (OPD) where history taking, physical examination and blood sampling took place. Children younger than 8 years of age performed symptom and peak flow registration during 1 week after the visit to the OPD. Results The number of eosinophils were significantly higher in children with asthma, compared with healthy controls. The concentration of IFNγ in supematants of cultures of stimulated PBMCs was significantly lower and the ratio of IL-4/IFNγ was significantly higher in children with asthma compared with healthy controls. The FEV₁ was directly and IgE was inversely related to the concentration of IFNγ in supematants of cultures of stimulated PBMCs. Conclusion IFNγ may play an important role in the pathophysiology of childhood atopic asthma.     

Interferon-gamma- and interleukin-4-producing T cells in Down's syndrome

Down's syndrome (DS) associates with genetic-dependent dysregulation of the interferon (IFN) system. We used intracellular cytokine staining to analyse the percentages of IFN-gamma- and interleukin (IL)-4-producing T cells in the peripheral blood of patients with DS, individuals with mental retardation (MR), and healthy controls (HCs). The percentages of IFN-gamma-producing CD4(+) and CD8(+) T cells (IFGCs), namely Th1 (mean, 21.4+/-S.D. 1.3) and Tc1 (12.6+/-1.1), and the Th1/Th2 ratio (6.1+/-0.2) in DS were significantly higher than in MR (15.9+/-1.3, 7.9+/-0.6, 4.8+/-0.3) and in HCs (15.6+/-1.9, 7.2+/-1.1, 4.6+/-0.6). Most of the DS patients with high IFGC percentages were seropositive for anti-transglutaminase IgA. We found no correlation between sex, age, APOE genotypes, coexisting autoimmune diseases, susceptibility to infections, or degree of cognitive impairment and high IFGC percentages. This abnormality might thus contribute to immune dysfunction in DS without manifest clinical correlates.     

Recurrent wheezing after respiratory syncytial virus or non-respiratory syncytial virus bronchiolitis in infancy: a 3-year follow-up

Background:  Recent studies have suggested that rhinovirus-associated early wheezing is a greater risk factor for development of recurrent wheezing in children than is early wheezing associated with respiratory syncytial virus (RSV). We determined the development of recurrent wheezing in young children within 3 years after hospitalization for RSV or non-RSV bronchiolitis.
Methods:  We identified retrospectively all children <2 years of age who were admitted to Turku University Hospital because of bronchiolitis in the months of August–December during 1988–2001. The primary outcome was recurrent wheezing that required long-term asthma medication. Data on asthma medications of the individual children were derived from the Social Insurance Institution of Finland.
Results:  Within the first year after hospitalization, 36 of 217 (16.6%) children with non-RSV bronchiolitis developed recurrent wheezing, compared with five of 199 (2.5%) children with RSV bronchiolitis [relative risk (RR) 6.6; 95% confidence interval (CI) 2.6–16.5]. The rates of recurrent wheezing were significantly increased in the non-RSV group also within 2 years (RR 2.9; 95% CI 1.7–5.1) and 3 years (RR 3.4; 95% CI 2.0–5.7) after hospitalization. The increased risk of recurrent wheezing in children with non-RSV-associated bronchiolitis was observed both in boys and girls at all time points of the 3-year follow-up, and it was not explained by the age difference between the RSV and non-RSV groups or any confounding seasonal factors.
Conclusion:  Children hospitalized with bronchiolitis caused by other viruses than RSV develop recurrent wheezing at substantially higher rates during a 3-year follow-up period than do children with RSV-induced bronchiolitis.     

Default development of cloned human naive CD4 T cells into interleukin-4- and interleukin-5-producing effector cells

It was recently demonstrated that naive human and mouse CD4 T cells release low but sufficient levels of interleukin (IL)-4 at priming to support their development into IL-4 producers. To determine whether this IL-4 is produced by a minor subset of cells, freshly isolated human naive CD4 T cells were directly cloned by limiting dilution in the absence of exogenous IL-4. More than 95% of neonatal and 60% of adult naive T cells seeded in single-cell cultures could be expanded upon stimulation with anti-CD3 mAb immobilized on CD32-B7.1 L cell transfectants in the presence of IL-2. All 171 clones derived from four neonates and two adults produced IL-4 and IL-5 at generally high levels. Like the allergen-specific human Th2 clones described in the literature, these T cell clones produced little or no interferon γ upon stimulation via their T cell receptor/CD3 complex, whereas they released high levels of this cytokine when activated with phorbol 12-myristate 13-acetate + ionomycin. Cells cloned and expanded in the presence of anti-IL4 + anti-IL-4R mAb produced much lower levels of IL-4 and IL-5. It is concluded that almost every single naive human CD4 T cell primed and expanded in the absence of exogenous IL-4 releases sufficient autocrine IL-4 to support its clonal expansion into high IL-4/IL-5 producers.