Allergen-stimulated interleukin-4 and interferon-gamma production in primary culture: responses of subjects with allergic rhinitis and normal controls.

The balance of interleukin-4 (IL-4) to interferon-gamma (IFN-gamma) production that is induced following exposure to common environmental antigens is believed to be instrumental in determining whether hypersensitivity or clinical unresponsiveness results to that antigen. To date, evaluation of cytokine (protein) production has been based predominantly on allergen-reactive CD4 T-cell clones or activation of fresh unselected peripheral blood mononuclear cell (PBMC) populations with non-physiological stimuli such as phorbal myristate acetate (PMA) and calcium ionophore, phytohaemagglutinin (PHA), anti-CD3 or anti-CD2/anti-CD28 monoclonal antibodies (mAb). Here, ultrasensitive IL-4 and IFN-gamma assays were optimized to allow direct analysis of antigen-stimulated cytokine production by fresh human PBMC. Primary cultures of cells from grass pollen-sensitive allergic rhinitis subjects and non-atopic controls were stimulated using a range of grass pollen allergen concentrations in the absence of exogenous cytokines or polyclonal activators. The majority of subjects (45 of 52) exhibited chloroquine-sensitive, CD4-dependent cytokine production in allergen-stimulated, short-term primary culture. Median IL-4 production was substantially greater among atopics (13.0 pg/ml versus < 1 pg/ml, Mann-Whitney U test, P < 0.0000001) and IFN-gamma was lower (P = 0.008), providing direct evidence for an imbalance in both IL-4 and IFN-gamma production among circulating, pollen-reactive cells in individuals with seasonal allergic rhinitis. The distinction in the allergen-driven cytokine responses elicited from normal and atopic donors was underscored by examination of the ratios of IFN-gamma:IL-4 synthesis. Non-atopic individuals exhibited intense IFN-gamma dominance of the T-cell response, in marked contrast to that observed among grass pollen-sensitive individuals (median IFN-gamma:IL-4 ratios of 14.0 versus 0.096, P = 0.000002). The observation that essentially all individuals produced IFN-gamma (+/- IL-4) following antigen stimulation in vitro argues that the most relevant consideration in determining susceptibility to immediate hypersensitivity versus clinical tolerance to environmental allergens is not a genetically defined capacity to recognize the antigen (i.e. if allergen-reactive T cells are present in that individual) but the nature of the cytokine response.     

Proliferation and release of IL-5 and IFN-gamma by peripheral blood mononuclear cells from cat-allergic asthmatics and rhinitics, non-cat-allergic asthmatics, and normal controls to peptides derived from Fel d 1 chain 1.

BACKGROUND: In general, T cells from normal, nonatopic individuals respond to aeroallergens with synthesis and release of IFN-gamma. In contrast, release of T(H)2-type cytokines by activated lymphocytes is a feature of allergic rhinitis and atopic asthma. OBJECTIVE: The purpose of this study was to determine differences in T-cell recognition of epitopes within allergenic sequences, in terms of proliferation and cytokine production, in subjects with atopic asthma compared with subjects with allergic rhinitis and normal controls. METHODS: Proliferative responses and IL-5/IFN-gamma release patterns from PBMCs from cat-allergic asthmatic, cat-allergic rhinitic, and non-cat-allergic asthmatic subjects and nonatopic normal controls were determined in primary cultures. Cells were challenged with 7 overlapping peptides spanning chain 1 of the major cat allergen, Fel d 1. RESULTS: The 4 groups did not differ with respect to the ability to mount proliferative responses to Fel d 1 peptides. In all groups, the IFN-gamma responses were predominantly to the amino terminus peptides. Cat-allergic and non-cat-allergic asthmatic subjects (and not cat-allergic rhinitic subjects and normal controls) made IL-5 responses to most of the Fel d 1 peptides, the result being a mixed (T(H)0) cytokine response at the N-terminus and a restricted (T(H)2) response at the C-terminus. CONCLUSION: Proliferative and IL-5/IFN-gamma responses of T cells from asthmatic and atopic rhinitic subjects and normal controls to allergen peptides can be dissociated. Furthermore, differing cytokine responses to peptides derived from a single antigen suggest that certain domains of the molecule might preferentially induce IL-5 rather than IFN-gamma and as a result could be more important in disease pathogenesis.     

In vitro responsiveness to IL-18 in combination with IL-12 or IL-2 by PBMC from patients with bronchial asthma and atopic dermatitis

Interleukin (IL)-18 is a proinflammatory cytokine and is now recognized as an important regulator of both helper T cells (Th) 1 and 2 cytokine production. An increased IL-18 secretion has been reported in patients with allergic disorders. It is predominantly produced by activated macrophages, and synergizes with IL-12 and IL-2 to induce IFN-gamma synthesis, thereby promoting Th1 cytokine response. Paradoxically, IL-18, by itself, strongly induces immunoglobulin (Ig) E and allergic inflammation, indicating a role for IL-18 in promoting Th2 response. We investigated the inducing effect in vitro of combining IL-18 and Il-12 or Il-2 on Th1- and Th2-type cytokines production by peripheral blood mononuclear cells (PBMC) from patients with allergic diseases. PBMC derived from 44 allergic patients [23 bronchial asthma (BA) and 21 atopic dermatitis (AD)] and 20 healthy controls were cultured with IL-18 in the presence of phytohemagglutinin (PHA) and IL-12 or IL-2. The levels of IFN-gamma, IL-13, and IL-4 in the culture supernatants were measured using enzymatic immunoassaying. IFN-gamma production was detected in all cultures from nonallergic controls stimulated with IL-18 in the presence of IL-12; however, the results for five BA patients and five AD patients were under the detection limit for IFN-gamma. In collaboration with IL-2, IL-18 was able to induce IFN-gamma production by PBMCs from all nonallergic controls and all allergic patients, with the exception of one AD patient. Synergistic induction of IL-13 production was found in cultures with IL-18 + IL-2, and the IL-13 induction was significantly increased in BA patients when compared with that in nonallergic controls (P = 0.006). The stimulation by IL-18, even in combination with IL-2, failed to induce IL-4 production by PBMC from both nonallergic controls and allergic patients. Although the induction of IFN-gamma by IL-18 + IL-12 was impaired in around a quarter of the allergic patients, the impairment of the IFN-gamma production was completely restored by IL-2 in the presence of IL-18. Thus, IL-18 enhances IFN-gamma production through an IL-12-dependent pathway and exhibits synergism when combined with IL-2 in terms of enhanced IL-13 and IFN-gamma production, suggesting the involvement of IL-18/IL-12/IL-2 pathway in modulating Th1/Th2 cytokine response.     

Allergen-specific production of interferon-γ by peripheral blood mononuclear cells and CD8 T cells in allergic disease and following immunotherapy

BACKGROUND: CD8 T cells are important immunoregulatory cells in animal models of allergic disease, but their role in human allergic immune responses has not been defined. With the development of novel immunotherapeutic reagents, it is clearly important to ascertain whether CD8 T-cell responses are altered following conventional allergen-specific immunotherapy (SIT) and hence targets for future developments/strategies. OBJECTIVE: To study the allergen-specific cytokine release of freshly isolated CD8 T cells from the blood of separate groups of house dust mite- (HDM) allergic patients, patients post-SIT and control nonatopic donors. METHODS: CD8 T cells were isolated by positive selection with immunomagnetic beads and cultured with the affinity purified major mite allergen Der p 1 or with different mitogens, using irradiated autologous peripheral blood mononuclear cells as antigen-presenting cells (APCs). Supernatants were collected at a number of time points and assayed by ELISA for the cytokines interleukin (IL) -4, IL-5 and interferon-gamma (IFNgamma). RESULTS: CD8 T cells stimulated with Der p 1 produced significant quantities of IFNgamma with cells from HDM-allergic subjects releasing considerably more IFNgamma than cells from nonatopic subjects, an average of 804 +/- 283 pg/mL of supernatant compared with 30.2 +/- 18.8 pg/mL (P = 0.006). The cytokine was detected in cultures of 16/17 of the allergic subjects and 4/7 of the nonatopic. CD8 T cells from 6/10 patients who had received HDM-SIT released IFNgamma at an average of 363 +/- 202 pg/mL, which was less than the allergic group but still higher than the nonatopic (P = 0.05). Equivalent levels of IFNgamma were detected when the cells were stimulated with the mitogen PHA and this was the same in all groups. Reliable allergen-specific release of significant quantities of IL-4 or IL-5 was not detected from CD8 T cells. CONCLUSION: Allergen-specific IFNgamma is produced at far greater levels from CD8 T cells of HDM-sensitive allergic patients than from nonatopic control individuals and this level is reduced following SIT.     

Aberrant interleukin (IL)-4 and IL-5 production in vitro by CD4+ helper T cells from atopic subjects.

The cytokine secretion profiles of T cell lines (TCL) specific for purified protein derivative (PPD) or streptokinase (SK), contemporarily derived from nine atopic and nine nonatopic individuals, were compared. Upon stimulation with phorbol myristate acetate (PMA) plus anti-CD3 monoclonal antibody (mAb), all TCL from both atopics and nonatopics produced interleukin (IL)-2 and interferon (IFN)-gamma. The mean IL-2 production by PPD- or SK-specific TCL from both atopics and nonatopics was similar, whereas the mean IFN-gamma production by TCL derived from atopics was significantly lower. In addition, both PPD- and SK-specific TCL from atopics produced detectable amounts of IL-4 and IL-5, whereas the corresponding TCL derived from nonatopics did not. A total number of 107 and 99 PPD-specific CD4+ T cell clones (TCC) were then derived from TCL of 4 atopic and 4 nonatopic donors and assessed for their profile of cytokine production in response to stimulation with either PMA plus anti-CD3 mAb or the specific antigen. Under both these experimental conditions, virtually all PPD-specific TCC from both atopic and nonatopic individuals produced IL-2 and IFN-gamma. In contrast, the great majority of PPD-specific TCC derived from nonatopic individuals did not produce IL-4 and IL-5, whereas high proportions of PPD-specific TCC derived from atopic donors displayed the ability to produce noticeable amounts of IL-4 and IL-5 besides IL-2 and IFN-gamma. These data indicate that CD4+ T cells from atopic individuals are able to produce IL-4 and IL-5 in response to bacterial antigens, such as PPD and SK, that usually evoke responses with a restricted type-1 T helper (Th1)-like cytokine profile in nonatopic individuals. Aberrant IL-4 production by Th cells may represent one of the immune alterations responsible for enhanced IgE antibody production in atopic people.     

Bacillus Calmette–Guérin-induced interleukin-12 did not additionally improve clinical and immunologic parameters in asthmatic children treated with sublingual immunotherapy

OBJECTIVE: To evaluate the effect of bacillus Calmette-Guérin (BCG) as an adjuvant to specific sublingual immunotherapy (SLIT) on the cytokine profile of peripheral blood mononuclear cells (PBMCs) and clinical outcome. METHODS: Thirty-two children with asthma and rhinitis allergic to house dust mite (HDM) with negative purified protein derivative (PPD) skin test response were enrolled. After a run-in period of 8 weeks, patients were randomized to receive either SLIT only (n=16) or one dose of BCG immunization before initiation of SLIT (n=16) with a standardized Dermatophagoides pteronyssinus (D. pteronyssinus)+D. farinea 50/50 extract. PPD-negative asthmatics (n=5) allergic to HDM receiving inhaled therapy only were included for comparison of cytokine levels in PBMC cultures. Efficacy was assessed both at the end of run-in and 6 months of treatment periods with criteria including symptom, medication and quality-of-life (QoL) scores, IgE levels, lung function, provocation concentration (PC20), eosinophil count and skin prick tests. IL-4, IL-5, IL-10, IL-12, IL-13 and IFN-gamma levels were determined in antigen specifically and polyclonally stimulated PBMC cultures. RESULTS: Both treatment groups showed significant improvement at the end of 6 months for asthma and rhinitis scores and QoL, number of asthma attacks, amount of beta2-agonists, inhaled and intranasal steroids, blood eosinophil counts and PC20. Interestingly, phytohaemagglutinin (PHA)-stimulated IL-12 and D. pteronyssinus-stimulated IFN-gamma in PBMC were significantly higher in the treatment groups than controls. In addition, IL-12 levels in response to D. pteronyssinus and PHA stimulation were significantly higher in the SLIT+BCG group than the SLIT alone group and controls. CONCLUSION: The present study demonstrates that successful SLIT is parallel to increased IFN-gamma production by PBMC. Although simultaneous BCG vaccination enhanced IL-12 production, it did not additionally improve the clinical outcome.     

Dendritic cells contribute to the development of atopy by an insufficiency in IL-12 production.

BACKGROUND: IL-12 is a crucial factor in the development and course of allergic diseases. By virtue of their IL-12 production, dendritic cells (DCs) are potent inducers of T(H)1 responses. However, distinct subsets of DCs have also been shown to induce T(H)2 differentiation. OBJECTIVE: We hypothesized that DCs from atopic and nonatopic individuals might differ in their propensity to skew T-cell responses to either the T(H)1 type or the T(H)2 type. To this end, we investigated the cytokine patterns produced by DCs from atopic and nonatopic individuals, and we attempted to clarify whether this could be due to different DC lineages or, alternatively, to different microenvironmental factors. METHODS: DCs were generated from lymphocyte-depleted PBMCs from atopic and nonatopic donors and fully matured with monocyte-conditioned medium. Production of IL-4, IL-5, IL-10, IL-12, and IL-13 in response to CD40 ligation was measured with ELISA. DC subsets were identified in PBMCs from freshly drawn blood by 3-color flow cytometry. RESULTS: Compared with DCs from healthy donors, monocyte-derived DCs from atopic patients produced less bioactive IL-12 and IL-10. DC production of IL-4, IL-13, and IL-5 was not detected. Relatively more CD123(+) DCs, corresponding to T(H)2-inducing "DC2s," were found in PBMCs from atopic patients. CONCLUSION: The data suggest that in addition to the described abnormalities in the patients' T-cell populations, DCs might also critically contribute to the atopic/allergic T(H)1 outcome in the patient and thus to the disease.     

Induction of IL-10+CD4+CD25+ T cells by grass pollen immunotherapy

BACKGROUND: Immunotherapy involves the modulation of allergen-specific T-cell responses, either T(H)2-to-T(H)1 immune deviation or, in bee venom-treated patients, induction of IL-10 production by CD4+CD25+ T cells. IL-10-producing CD4+CD25+ regulatory T cells have emerged as potential mediators of immune tolerance in numerous murine models of immunopathology. OBJECTIVE: The aim of this study was to evaluate the role of IL-10 production and CD4+CD25+ T cells in the response to grass pollen immunotherapy. METHODS: PBMCs were isolated from patients after 1 year of grass pollen immunotherapy and from matched untreated atopic and healthy control subjects. After 6 days of in vitro stimulation with Phleum pratense, production of IL-10, IL-5, IL-4, and IFN-gamma and proliferation and numbers of CD4+CD25+ T cells were measured. T cells were then stimulated for a further 5 hours with phorbol 12-myristate 13-acetate and ionomycin and assessed for intracellular IL-10 by means of flow cytometry. RESULTS: Patients undergoing immunotherapy produced significantly more IL-10 than atopic control subjects (patients undergoing immunotherapy, 116 +/- 21 pg/mL [n = 11]; atopic patients, 30 +/- 5 pg/mL [n = 11]; P <.001), and the number of CD4+CD25+ cells identified after allergen stimulation was also greater in the immunotherapy group. The numbers of CD4+CD25+ T cells correlated positively with activation as measured by proliferation in both of the control groups but not in the immunotherapy group. Moreover, only T cells from patients undergoing immunotherapy were positive for intracellular IL-10, and these were almost exclusively CD4+CD25+ cells. CONCLUSION: Grass pollen immunotherapy results in a population of circulating T cells that express the IL-10(+) CD4+CD25+ phenotype in response to allergen restimulation.     

IL-13 production by allergen-stimulated T cells is increased in allergic disease and associated with IL-5 but not IFN-gamma expression.

Interleukin-13 (IL-13) shares many, but not all, of the properties of the prototypic T-helper type 2 (Th2) cytokine IL-4, but its role in allergen-driven T-cell responses remains poorly defined. We hypothesized that allergen stimulation of peripheral blood T cells from patients with atopic disease compared with non-atopic controls results in elevated IL-13 synthesis in the context of a 'Th2-type' pattern. Freshly isolated peripheral blood mononuclear cells (PBMC) obtained from sensitized atopic patients with allergic disease, and non-atopic control subjects, were cultured with the allergens Phleum pratense (Timothy grass pollen) or Dermatophagoides pteronyssinus (house dust mite) and the non-allergenic recall antigen Mycobacterium tuberculosis purified protein derivative (PPD). Supernatant concentrations of IL-13, along with IL-5 and interferon-gamma (IFN-gamma) (Th2- and Th1-type cytokines, respectively) were determined by enzyme-linked immunosorbent assay (ELISA). Allergen-induced IL-13 and IL-5 production by T cells from patients with allergic disease was markedly elevated (P = 0.0075 and P = 0.0004, respectively) compared with non-atopic controls, whereas IFN-gamma production was not significantly different. In contrast to allergen, the prototypic Th1-type antigen M. tuberculosis PPD induced an excess of IFN-gamma over IL-13 and IL-5 production, and absolute concentrations of cytokines were not affected by the presence or absence of atopic disease. Addition of exogenous recombinant IFN-gamma or IL-12, cytokines known to inhibit Th2-type responses, significantly inhibited allergen-driven production of both IL-13 and IL-5, but not T-cell proliferation, whereas exogenous IL-4 did not significantly affect production of IL-13 or IL-5. We conclude that allergen-specific T cells from atopic subjects secrete elevated quantities of IL-13 compared with non-atopic controls, in the context of a Th2-type pattern of cytokine production.     

Intracellular IL-5 and T-lymphocyte subsets in atopic and nonatopic bronchial asthma

BACKGROUND: Allergen-stimulated IL-5 production by CD4+ T cells is the key issue in atopic asthma. On the other hand, virus-specific CD8+ T cells produce IL-5 and might play an important role in the pathogenesis of nonatopic asthma. OBJECTIVES: We sought to compare the IL-5-producing T-lymphocyte subsets in the peripheral blood of atopic and nonatopic asthmatic subjects, especially the contribution of IL-5-producing CD8(+) T cells in nonatopic asthma. METHODS: Heparinized blood samples were obtained from subjects with atopic asthma (n = 10), subjects with nonatopic asthma (n = 10), and healthy subjects (n = 10) and stimulated with ionomycin and phorbol myristate acetate in the presence of brefeldin A. Two-color flow cytometric analysis with mAbs to cell-surface antigens and intracellular IL-5 was used to detect the IL-5-producing T-cell subsets. RESULTS: A higher percentage of IL-5-producing CD3(+) T cells was detected in subjects with atopic and nonatopic asthma than that seen in the healthy subjects. The percentage of IL-5-producing CD4(+) T cells was significantly higher in subjects with atopic asthma than in the healthy subjects. The percentage of IL-5-producing CD8(+) T cells was significantly higher in subjects with nonatopic asthma than in the healthy subjects. The percentage of IL-5-producing CD8(+) T cells was higher in subjects with nonatopic asthma than in those with atopic asthma, but the difference was not statistically significant. CONCLUSIONS: CD4(+) T cells are the major source of IL-5 among CD3(+) lymphocytes in subjects with atopic asthma. On the other hand, increased IL-5 production by CD8(+) T cells, as well as by CD4(+) T cells, is a characteristic feature of nonatopic asthma.     

The profiles of interleukin (IL)-2, IL-6, and interferon-gamma production by peripheral blood mononuclear cells from house-dust-mite-allergic patients: a role for IL-6 in allergic disease

We have developed a model to measure cytokine production by peripheral blood mononuclear cells (PBMC) in vitro. In this report, we examine the production of interleukin-2 (IL-2), IL-6, and interferon-gamma (IFN-γ) by PBMC of house-dust-mite ( Dermatophagoides pteronyssinus )-allergic subjects. When stimulated with specific allergen ( D. pteronyssinus ), PBMC of patients produced significant levels of IL-2 and high levels of IL-6, but little or no IFN-γ. Nonatopic control PBMC also produced IL-6, although at lower levels, but no IL-2 or IFN-γ. A ubiquitous antigen, streptokinase/streptodornase (SKSD), induced high levels of IL-2 in patients, but only low levels of IFN-γ and IL-6. Nonatopic controls produced similar levels of IL-2 and IL-6, but high levels of IFN-γ to SKSD. IL-2 and IFN-γ levels induced by the T-cell mitogen phytohaemagglutinin (PHA) were similar in patient and control groups, but IL-6 levels were significantly lower in the patients. IgE synthesis in vitro was shown only in atopic PBMC cultures stimulated with specific allergen. The major points can be summarized as 1) IL-2 production by atopic patients in response to allergen; 2) IL-6 production to allergen by both atopic and nonatopic patients, but significantly increased in atopic patients; and 3) defective IFN-γ production by atopic patients to both allergen and antigen. These findings suggest that IL-6 may be important in the immune response to inhalent allergens such as D. pteronyssinus , possibly by creating a cytokine environment favourable to a T^H2 response, and that atopic patients exhibit a generalized defect of IFN-γ production, not related to the response to allergen.     

Enhanced ELISPOT detection of antigen-specific T cell responses from cryopreserved specimens with addition of both IL-7 and IL-15--the Amplispot assay

The importance of the enzyme-linked immunosorbent spot (ELISPOT) assay as a tool for studying immune responses in vitro is becoming increasingly apparent. However, there remains a need for enhanced sensitivity for the detection of low frequency antigen-specific T cell responses. We reasoned that the addition of a combination of the cytokines interleukin (IL)-7 and IL-15 would selectively increase interferon-gamma (IFN-gamma) production from antigen-stimulated CD4+ and CD8+ effector memory T cells. Freshly isolated or cryopreserved peripheral blood mononuclear cells (PBMC) from four healthy donors were analysed by ELISPOT for the frequency of purified protein derivative (PPD)-specific CD4+ T cells or cytomegalovirus (CMV) peptide-specific CD8+ T cells. Addition of IL-7 and IL-15 increased the number of PPD-specific CD4+ T cells up to 2.4-fold in fresh PBMC and up to 18-fold in cryopreserved PBMC. The cytokines also increased the number of CMV peptide-specific CD8+ T cells in fresh PBMC up to 7.5-fold. No additional increases were seen when antibodies to co-stimulatory molecules CD28 and CD49d were applied together with the cytokine combination. These data demonstrate that the sensitivity of the ELISPOT assay may be significantly augmented by addition of the cytokines IL-7 and IL-15 to antigen-stimulated cells. This method will be particularly useful for the assessment of antigen-stimulated cytokine production by T cells in cryopreserved biological specimens.     

Diminished IL-10 production in subjects with allergy after infection with influenza A virus

BACKGROUND: Recent studies have documented a link between respiratory viral infections and the expression of asthma and other allergic disorders. Results from other studies have suggested that diminished production of IL-10, an anti-inflammatory cytokine, may contribute to the pathophysiologic features of these diseases. OBJECTIVE: The objective of this study was to determine whether diminished IL-10 production and TH2 cytokine skewing occur in allergic, as compared with nonallergic, subjects after experimental infection with the influenza A virus. METHODS: PBMCs were isolated from 11 subjects with allergy and 14 subjects with no allergy before and after influenza A infection and stimulated with either mitogen (PHA) or antigen (influenza A). Supernatants were assayed for IL-10, IL-4, and IFN-gamma by ELISA. RESULTS: PBMC IL-10 production was significantly diminished in subjects with allergy, as compared with subjects with no allergy, after experimental infection with influenza A virus. However, significant TH2 skewing and enhanced airway symptoms were not observed in these same subjects. CONCLUSIONS: These data provide further support that subjects with allergy have an intrinsic inability to upregulate IL-10 production in response to inflammatory stimuli and extend this observation to include respiratory viral infections. Future studies in this area could lead to a better understanding of the pathogenesis of asthma and other allergic disorders     

PHA-induced IL-12Rβ2 mRNA expression in atopic and non-atopic children

BACKGROUND: IL-12 is a strong inducer of Th1 responses. Stimulation via the CD2 receptor increases IFN-gamma production and enhances the responsiveness of activated T-cells to IL-12, possibly due to an up-regulation of the signal transducing beta(2) chain of the IL-12 receptor (IL-12R beta(2)). Atopic children have a reduced Th1-like immunity and a reduced CD2 expression. Our hypothesis is that atopic individuals have a reduced function of the CD2 pathway, causing reduced responsiveness to IL-12 and decreased IFN-gamma production. OBJECTIVE: The aim was to study the mRNA expression of the IL-12R beta(2) chain, after stimulation via the CD2 pathway in peripheral blood mononuclear cells (PBMC), of atopic and non-atopic children, and to investigate correlations to the production of Th1 and Th2 cytokines. MATERIALS AND METHODS: The study included 23 skin prick test positive, and 9 non-sensitized, 12-year-old children. PBMC were stimulated for 24 h with phytohemagglutinin (PHA) (2 microg/mL), which stimulates T cells through the CD2 pathway. Expression of IL-12R beta(2) mRNA was analysed by quantitative real time PCR and the cytokine production was detected with ELISA. RESULTS: Atopic and non-atopic children had similar baseline expression of IL-12R beta(2) mRNA, whereas PHA-induced IL-12R beta(2) mRNA expression was lower in atopic than in non-atopic children. The PHA-induced IL-12R beta(2) mRNA expression correlated well with the PHA-induced IFN-gamma production and with the IFN-gamma/IL-4 ratio. CONCLUSION: PBMC from atopic children expressed less IL-12R beta(2) mRNA than non-atopic children after stimulation via the CD2 pathway (PHA). This may indicate a reduced capacity to respond to Th1-inducing stimuli in atopic children.     

Study of the Th1/Th2 balance, including IL-10 production, in cultures of peripheral blood mononuclear cells from birch-pollen-allergic patients

BACKGROUND: Excessive production of interleukin (IL)-4, IL-5, IL-10, and IL-13 is thought to be important in the development of allergy and asthma. The objective of this investigation was to study Th1/Th2-like cytokine profiles in vitro in seven patients allergic to birch pollen and six nonallergic controls during the birch-pollen season. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated and cultured with birch-pollen extract (BPE) or tetanus toxoid (TT) for 7 days, harvested, and restimulated with the mitogens phytohemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA) for 24 h. Cytokine production was determined by ELISA, and logarithmic cytokine ratios were compared between the two groups and between the antigens. RESULTS: In the allergic group, the cultures prestimulated with BPE had a more Th2-like cytokine response than the TT-prestimulated cultures; i.e., lower IFN-gamma and higher IL-10 production (P<0.05), as well as higher IL-5/IFN-gamma and IL-13/ IFN-gamma ratios (P<0.05). There were also significantly higher IL-4/IFN-gamma (P<0.005) and IL-5/IFN-gamma (P<0.05) ratios in BPE-stimulated cultures in the allergic group than in the control group. The IL-4 and IL-13 production in vitro correlated with the specific serum IgE levels. CONCLUSIONS: BPE stimulation induces a Th2-like cytokine response by PBMC isolated during the pollen season from birch-pollen-allergic patients, indicating a Th2-type immune response to birch pollen in vivo.     

Parental tobacco smoking is associated with augmented IL-13 secretion in children with allergic asthma

BACKGROUND: Exposure to environmental tobacco smoke (ETS) has been shown to increase symptoms of allergic bronchial asthma, but direct effects on the expression of inflammatory markers have not been demonstrated thus far. OBJECTIVE: The aim of this study was to assess the correlation of ETS exposure with the expression of proinflammatory mediators in airway secretions, including IFN-gamma and IL-12, as well as IL-5 and IL-13, in allergic asthmatic schoolchildren and healthy control subjects. METHODS: By using the nasopharyngeal aspiration technique, airway secretions were collected from 24 atopic children with asthma (age, 6-16 years) and 26 healthy control subjects, and the concentration of cytokines was measured with immunoenzymatic methods. RESULTS: IL-13 levels were highly increased in patients with asthma (P < .005), and parental tobacco smoke resulted in a significant increase in airway IL-13 secretion in these children compared with that seen in nonexposed children and healthy control subjects (median, 860 pg/mL vs 242 pg/mL and 125 pg/mL, respectively). Furthermore, a positive correlation between IL-13 levels and serum IgE concentrations (r(s) = 0.55) was found in children with allergic asthma. CONCLUSIONS: These results indicate that ETS augments the expression and secretion of IL-13 in allergic asthma and that nasopharyngeal aspiration is a suitable method to assess cytokine measurements in airways in children. Measurements of IL-13 in secretions might be taken into account as a noninvasive marker of airway inflammation and to assess the detrimental effects of ETS.     

Increased sensitivity to IL-4 in patients with allergic bronchopulmonary aspergillosis

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is characterized by a heightened Th2 CD4+ cell response to Aspergillus fumigatus allergens and a hyper-IgE state compared to atopic asthmatic and cystic fibrosis patients without ABPA. We hypothesized that one reason for this response is increased sensitivity to IL-4 in ABPA, resulting in increased expression of CD23 and CD86, leading to a positive amplification mechanism which increases Th2 CD4+ T cell responses. METHODS: Peripheral blood mononuclear cells isolated from 10 ABPA, 9 atopic, and 8 nonatopic subjects and stimulated for 48 h with varying concentrations of rIL-4 ranging from 0.1 to 50 ng/ml. The percentages of CD23+ and CD86+ B cells and the number of CD23+ molecules on CD20+ and CD86+CD20+ B cells were quantified by flow cytometry. RESULTS: Total serum IgE levels were elevated in ABPA patients compared to atopic and nonatopic controls. At day 0 prior to culture, CD23 molecules per CD20+ B cell were significantly elevated in ABPA patients compared to atopic and to nonatopic patients. CD23 molecules per CD20+ B cell in ABPA and atopic patients decreased after 48 h in culture without IL-4 added and were similar. With IL-4 stimulation, ABPA patients had significantly increased rates of CD23 expression per B cell compared to atopic and nonatopic subjects (p < 0.001). Furthermore, ABPA had significantly increased numbers of CD23+ molecules per B cell and CD86+ B cell following IL-4 stimulation compared to atopic and nonatopic patients. Both ABPA and atopic patients at day 0 prior to culture had increased expression of CD86+ and CD23+CD86+ B cells compared to nonatopic patients. After 48 h in culture without IL-4, the percentages of CD86+ and CD23+CD86+ B cells decreased in ABPA and atopic patients. After stimulation with IL-4, ABPA patients had significant upregulation of CD23+CD86+ B cells compared to atopic and nonatopic patients. Similarly, the number of CD23 molecules per CD86+CD20+ B cell was significantly upregulated following IL-4 stimulation in ABPA patients compared to atopic and to nonatopic subjects. CONCLUSIONS: This is the first study to demonstrate that ABPA patients have increased sensitivity to IL-4 stimulation compared to other atopic individuals, such that ABPA > atopic > nonatopic patients. The B cells from ABPA patients were significantly more sensitive to IL-4 stimulation compared to atopic and nonatopic patients with upregulation of CD23 and CD86 expression. ABPA subjects had increased CD86+ and CD23+CD86+ B cell expression on day 0 prior to culture and with upregulation of CD23+ molecules on CD86+CD20+ B cells. IL-4 also stimulated upregulated CD86+ expression on B cells in atopic patients with little effect on nonatopic patients. This study supports the premise that IL-4, IL-4R alpha and CD86 are central targets in the treatment of ABPA and atopic disease.