Sputum IgE and Cytokines in Asthma: Relationship with Sputum Cellular Profile

Background

Local IgE production may play a role in asthma pathogenesis. The aim of the study was to assess sputum total IgE and cytokines in asthmatics according to sputum cellular phenotype.

Methods

We studied 122 subjects including 22 non atopic healthy subjects, 41 eosinophilic (sputum eosinophils ≥3%), 16 neutrophilic (sputum neutrophils >76%) and 43 pauci-granulocytic asthmatics (sputum eosinophils <3% and sputum neutrophils ≤76%) recruited from the asthma clinic at CHU Liege.Sputum supernatant total IgE (tIgE) was measured by ImmunoCAP and sputum supernatant cytokines (IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IFN-γ and TNF-α) were measured with the Luminex xMAP Technology by using commercially available Fluorokine MAP kits.

Results

After concentrating sputum samples, total IgE was detectable in the majority of subjects. Sputum IgE was raised in asthmatics when compared to healthy subjects. Overall, asthmatics did not significantly differ from healthy subjects with respect to cytokine levels. The eosinophilic asthma phenotype, however, was characterised by raised sputum tIgE, IL-5 and IL-13 compared to healthy subjects (p<0.001, p<0.001 and p<0.05 respectively) and pauci-granulocytic asthma (p<0.01, p<0.001 and p<0.05 respectively) and raised IL-5 compared to neutrophilic asthma (p<0.01). When patients were classified according to sputum IgE levels, it appeared that IL-5, IL-6, IL-17 and TNF-α sputum supernatant levels were raised in the “IgE high” asthmatics (IgE ≥0.1 kU/l) when compared to “IgE low” asthmatics (IgE<0.1 kU/l).

Conclusion

The eosinophilic asthma phenotype was associated with raised sputum IgE and a Th2 cytokine profile. Raised sputum IgE was associated with a heterogeneous cytokine overproduction.     

Association between genes encoding components of the IL-4/IL-4 receptor pathway and dermatitis in children

Objective

To determine whether IL-4, IL-4Rα and STAT6 polymorphisms are associated with susceptibility to dermatitis in Egyptian children.

Methods

We genotyped three groups of children, consisting of 106 atopic dermatitis (AD) children, 95 non-AD children, and 100 of healthy controls, for IL-4 (− 590 C/T), (− 33 C/T), IL-4Rα (I50V), (Q576R) and STAT6 (2964 G/A), (2892 C/T) gene polymorphisms using PCR-RFLP assay. Total serum IgE and serum IL-4 levels were detected by ELISA.

Results

There was a non-significant association of IL-4 − 590 C/T, − 33 C/T polymorphisms in the children with non-AD or those with AD when compared with the controls. We identified a significant association between IL-4Rα I50V, Q576R polymorphisms and dermatitis susceptibility in AD (p = 0.002, < 0.001 respectively), whereas no such association was observed in non-AD group (p = 0.52, 0.99 respectively). A significant association between STAT6 polymorphisms and both types of dermatitis was found. Patients who were carriers of IL4 − 590C, IL-4Rα I50V G, STAT6 2964 A and STAT6 2892 T had an increased risk of AD [OR and 95% CI: 3.2 (2.5–4.2), p = 0.005]. Furthermore, there was no relation between each polymorphism and serum IL-4 level (p > 0.05 for each) while homozygosity for the risk alleles of IL-4, IL-4Rα and STAT6 SNPs were significantly associated with increased total IgE levels in all subjects.

Conclusion

In Egyptian children, the IL-4Rα and the STAT6 polymorphism may play a role in susceptibility to AD. In addition, gene–gene interaction between the IL-4, the IL-4Rα and the STAT6 significantly increases an individual's susceptibility to AD.     

Membranous glomerulonephritis development with Th2-type immune deviations in MRL/lpr mice deficient for IL-27 receptor (WSX-1)

MRL/lpr mice develop spontaneous glomerulonephritis that is essentially identical with diffuse proliferative glomerulonephritis (World Health Organization class IV) in human lupus nephritis. Lupus nephritis is one of the most serious complications of systemic lupus erythematosus. Diffuse proliferative glomerulonephritis is associated with autoimmune responses dominated by Th1 cells producing high levels of IFN-gamma. The initial mounting of Th1 responses depends on the function of the WSX-1 gene, which encodes a subunit of the IL-27R with homology to IL-12R. In mice deficient for the WSX-1 gene, proper Th1 differentiation was impaired and abnormal Th2 skewing was observed during infection with some intracellular pathogens. Disruption of the WSX-1 gene dramatically changed the pathophysiology of glomerulonephritis developing in MRL/lpr mice. WSX-1-/- MRL/lpr mice developed disease resembling human membranous glomerulonephritis (World Health Organization class V) with a predominance of IgG1 in glomerular deposits, accompanied by increased IgG1 and IgE in the sera. T cells in WSX-1-/- MRL/lpr mice displayed significantly reduced IFN-gamma production along with elevated IL-4 expression. Loss of WSX-1 thus favors Th2-type autoimmune responses, suggesting that the Th1/Th2 balance may be a pivotal determinant of human lupus nephritis development.     

A role for IL-12 receptor expression and signal transduction in host defense in leprosy.

The generation of cell-mediated immunity against intracellular infection involves the production of IL-12, a critical cytokine required for the development of Th1 responses. The biologic activities of IL-12 are mediated through a specific, high affinity IL-12R composed of an IL-12Rbeta1/IL-12Rbeta2 heterodimer, with the IL-12Rbeta2 chain involved in signaling via Stat4. We investigated IL-12R expression and function in human infectious disease, using the clinical/immunologic spectrum of leprosy as a model. T cells from tuberculoid patients, the resistant form of leprosy, are responsive to IL-12; however, T cells from lepromatous patients, the susceptible form of leprosy, do not respond to IL-12. We found that the IL-12Rbeta2 was more highly expressed in tuberculoid lesions compared with lepromatous lesions. In contrast, IL-12Rbeta1 expression was similar in both tuberculoid and lepromatous lesions. The expression of IL-12Rbeta2 on T cells was up-regulated by Mycobacterium leprae in tuberculoid but not in lepromatous patients. Furthermore, IL-12 induced Stat4 phosphorylation and DNA binding in M. leprae-activated T cells from tuberculoid but not from lepromatous patients. Interestingly, IL-12Rbeta2 in lepromatous patients could be up-regulated by stimulation with M. tuberculosis. These data suggest that Th response to M. leprae determines IL-12Rbeta2 expression and function in host defense in leprosy.     

Strain variation in the susceptibility and immune response to Clonorchis sinensis infection in mice

Mice have shown various susceptibility to infection by Clonorchis sinensis. To compare the intra-specific variation in the host-parasite relationship of C. sinensis, 6 strains of mice (ICR, BALB/c, C57BL/6, DDY, CBA/N, and C3H/HeN) with 3 different haplotypes were evaluated on their susceptibility. The worm recovery rate and immunological responses were observed after 4 and 8 weeks of infection with 30 metacercariae. The highest worm recovery rate was observed as 20.7% in the C3H/HeN strain after 4 weeks of infection along with histopathological changes. The rate was 10.0% in C57BL/6 mice after 8 weeks. ICR, BALB/c, and CBA/N showed elevated levels of IgE at both time points when compared to the rest of the strains. The serum IgG1 and IgG2a levels were elevated in most of the strains; however, the C57BL/6 strain showed a lower level of IgG2a that indicated the IgG1 predominance over IgG2a. The production of IL-4 after concanavalin-A stimulation of splenocytes slightly increased among the mouse strains except C3H/HeN after 4 or 8 weeks of infection, but each strain produced high levels of IFN-γ after 8 weeks, which implied mixed Th1/Th2 responses. ICR, DDY, CBA/N, and C3H/HeN strains showed a significantly increased level of IL-10 after 8 weeks as compared to C57BL/6. All of the strains showed an increased level of IL-13 and suggested fibrotic changes in the mice. In conclusion, mice are insusceptible to infection with C. sinensis; however, the C57BL/6, BALB/c and ICR strains are relatively susceptible after 8 weeks of infection among the six strains. Worm expulsion may be one of the causes of low susceptibility of C3H/HeN mice strain at the 8th week. Elevated IgE, IFN-γ, and IL-13 of infected mice suggest both Th1 and Th2 responses that may be related to the low host susceptibility.     

The Characteristics of Th1/Th2 Cytokine Receptors on Monocytes in Untreated Patients of Long Term Nonprogressor or Chronic HIV Infection

Monocytes/macrophages play crucial roles in immunity to microorganisms and are one of the important targets for human immunodeficiency virus (HIV) infection. The phenotypes and function of monocytes in HIV-infected patients were poorly determined. We herein detected the expression of Th1/Th2 cytokine receptors on monocyte subsets in the untreated HIV-infected patients of either long term nonprogressor (LTNP) or chronic infection (CHI). CD14+CD16- monocytes were significantly increased and CD14+CD16+ monocytes were reduced in patients of LTNP or CHI compared with healthy control. IL-6R expression on CD14+CD16- monocytes were decreased in patients of LTNP or CHI, whereas IL-4R and IL-10R expression on both CD14+CD16- and CD14+CD16+ monocyte subsets were increased in patients with LTNP or CHI, as determined by flow cytometry and real time PCR assays. The decreased IL-6R expression and enhanced IL-4R and IL-10R expression were also observed on CD4+ T cells of these patients, indicating that these changes in monocytes are not cell-specific. CD14+CD16- monocytes of HIV-infected patients produced less TNF-α and IL-1β but identical levels of IL-6, and IL-12 as the control after IFN-γ/LPS stimulation. However, in the presence of IL-4 or IL10, CD14+CD16- monocytes of HIV-infected patients produced more TNF-α, IL-6, IL-12 or Il-1β after IFN-γ/LPS stimulation than the healthy control, supporting the impaired IL-4R and IL-10R signal pathways in patients with LTNP and CHI. Therefore, our present study offered the basic information for the Th1/Th2 cytokine receptor expression and function on monocyte subsets in untreated HIV-infected individuals.     

An increased proportion of circulating Th22 and Tc22 cells in psoriasis

Psoriasis is a common dermatosis mediated by T cells. This study investigated the correlation of Th22 cells and Tc22 cells with psoriasis. A total of 30 psoriasis patients and 11 age- and sex-matched healthy controls were recruited for this study. The proportions of circulating Th22 and Tc22 cells, expression of aryl hydrocarbon receptor, and IL-22 levels in the psoriasis patients were significantly higher than those in the control subjects (p < 0.05). There was a positive correlation between the proportion of circulating Th22 cells, IL-22 levels, and PASI score. The IL-22 levels and PASI score were also positively correlated. There was no correlation between the proportion of circulating Tc22 cells and IL-22 level or PASI score. These data are consistent with Th22 cells involvement in the pathogenesis of psoriasis.     

Cutting edge: ectopic expression of the IL-12 receptor-beta 2 in developing and committed Th2 cells does not affect the production of IL-4 or induce the production of IFN-gamma

The IL-12 receptor-beta 2 (IL-12R beta 2) chain is expressed on Th1 cells and lost upon differentiation to the Th2 phenotype. This has been suggested as the basis for commitment of Th1 cells, because early differentiated Th2 cells do not reverse their phenotype and do not produce IFN-gamma on restimulation in the presence of IL-12. In this study, we ectopically expressed the IL-12 receptor-beta 2 (IL-12R beta 2) bicistronically with enhanced green fluorescent protein by retroviral infection in developing and committed Th2 cells. Restimulation of Th2 cells expressing this ectopic IL-12R beta 2 in the presence of IL-12 led to levels of IL-4 production similar to those in control Th2 cells. The expression of IL-12R beta 2 in Th2 cells did not lead to significant levels of IFN-gamma production, although IL-12-mediated STAT signaling and proliferation were restored. Thus, although the IL-12R beta 2 and IL-12-dependent STAT4 activation are required for Th1 responses, activation of this pathway is not sufficient to restore a Th1 phenotype in developing or committed Th2 cells.     

Activated STAT4 has an essential role in Th1 differentiation and proliferation that is independent of its role in the maintenance of IL-12R beta 2 chain expression and signaling

In this study we demonstrated that CD4(+) T cells from STAT4(-/-) mice exhibit reduced IL-12R expression and poor IL-12R signaling function. This raised the question of whether activated STAT4 participates in Th1 cell development mainly through its effects on IL-12 signaling. In a first approach to this question we determined the capacity of CD4(+) T cells from STAT4(-/-) bearing an IL-12Rbeta2 chain transgene (and thus capable of normal IL-12R expression and signaling) to undergo Th1 differentiation when stimulated by Con A and APCs. We found that such cells were still unable to exhibit IL-12-mediated IFN-gamma production. In a second approach to this question, we created Th2 cell lines (D10 cells) transfected with STAT4-expressing plasmids with various tyrosine-->phenylalanine mutations and CD4(+) T cell lines from IL-12beta2(-/-) mice infected with retroviruses expressing similarly STAT4 mutations that nevertheless express surface IL-12Rbeta2 chains. We then showed that constructs that were unable to support STAT4 tyrosine phosphorylation (in D10 cells) as a result of mutation were also incapable of supporting IL-12-induced IFN-gamma production (in IL-12Rbeta2(-/-) cells). Thus, by two complementary approaches we demonstrated that activated STAT4 has an essential downstream role in Th1 cell differentiation that is independent of its role in the support of IL-12Rbeta2 chain signaling. This implies that STAT4 is an essential element in the early events of Th1 differentiation.     

The association between IFN-γ and IL-4 genetic polymorphisms and childhood susceptibility to bronchial asthma

The present study aims to investigate the association between the genetic polymorphisms of interferon (IFN)-γ and interleukin (IL)-4 with childhood susceptibility to asthma and the levels of IFN-γ, IL-4, and immunoglobulin (Ig) E among asthmatic children. A total of 100 asthmatic children and 122 control children were enrolled in the present study. The genotypes of the IFN-γ gene at the − 179G/T locus and the IL-4 gene at the − 33C/T and − 589C/T loci were detected using polymerase chain reaction with restriction fragment length polymorphism. The IFN-γ gene at the + 874A/T locus and the IFN-γ CA repeats were tested using allele-specific and capillary electrophoresis, respectively, whereas the IFN-γ, IL-4, and total IgE levels were measured using enzyme-linked immunosorbent assays. The 100 asthmatic children and the 122 control children were all GG homozygous in the − 179 locus of the IFN-γ gene, which shows that the IFN-γ gene is not mutated at the − 179 locus. No significant differences were found in terms of genotypic and allelic frequency distribution in the IFN-γ gene or the CA repeat at the + 874A/T locus between the asthmatic children and the control (P > 0.05). An association was found between the polymorphism of the IFN-γ gene at + 874A/T and IFN-γ levels. IFN-γ expression was lower among patients with the AA genotype than those with the AT genotype (P < 0.05); the genotypic and allelic frequency distributions of the IL-4 gene at − 33C/T and − 589C/T were significantly different between the asthmatic children and the control (P < 0.05). The levels of IL-4 and IgE among children with TT genotype at the − 33 and − 589 loci were higher than those with the CT genotype, but only the polymorphism at − 33C/T was associated with IL-4 levels (P < 0.05). The polymorphisms of the IFN-γ gene at + 874A/T or the CA repeats are not correlated with susceptibility to asthma. Thus, the polymorphism at + 874A/T is correlated with IFN-γ level. The TT genotypes of the IL-4 gene at the − 33 and − 589 loci are associated with asthma susceptibility in children, and polymorphism at the − 33 locus may be associated with IL-4 level.     

Allergen rush immunotherapy increases interleukin (IL)-12 production and IL-12 receptor beta2 chain expression in patients with allergic asthma

Interleukin (IL)-12 production and IL-12 receptor (IL-12R) beta2 chain expression were investigated in patients with allergic asthma successfully treated with rush immunotherapy (RIT) and control patients with mild allergic asthma. Peripheral blood mononuclear cells (PBMCs) were stimulated with Dermatophagoides farinae (Der f), and production of cytokines was measured. Furthermore, the effects of cytokines on IL-12R beta2 chain expression on CD4(+) T cells were investigated. Production by PBMCs of IL-12 and IFN-gamma was significantly higher and production of IL-4 was significantly lower after stimulation with Der f allergen in RIT-treated patients than in control patients. Significant increases in the expression of IL-12R beta2 chain before and after stimulation of CD4(+) T cells with IL-12 or IFN-gamma were observed in RIT-treated patients compared with that in control patients. Allergen RIT increases IL-12 production and IL-12R beta2 chain expression and thus may convert cytokine production from Th2 to Th1 or Th0 type in allergic asthma.     

The mechanism of a defective IFN-gamma response to bacterial toxins in an atopic dermatitis model, NC/Nga mice, and the therapeutic effect of IFN-gamma, IL-12, or IL-18 on dermatitis

NC/Nga (NC) mice raised under conventional conditions (Conv. NC mice) spontaneously develop dermatitis similar to human atopic dermatitis, whereas NC mice raised under the specific pathogen-free conditions do not develop dermatitis. In the present study, we show that the representative Th1 cytokine, IFN-gamma levels in the sera of NC mice, injected with either staphylococcal enterotoxin B or endotoxin (LPS), to be severalfold lower than those of normal mice. The low IFN-gamma response to staphylococcal enterotoxin B was correlated to the lack of regular Vbeta8(+) T cells and Vbeta8(+) NK T cells, and the low IFN-gamma response to LPS was correlated to an impaired IL-18 production of macrophages. The CD3-stimulated IL-4 production from liver and spleen T cells from Conv. NC mice in vitro was greatly augmented. The serum IL-4 levels of untreated Conv. NC mice also were higher than those of normal mice and specific pathogen-free NC mice. Treatment of Conv. NC mice either with IFN-gamma, IL-12, or IL-18 twice a week from 4 wk of age substantially inhibited the elevation of the serum IgE levels, serum IL-4 levels, and dermatitis, and IL-12 or IL-18 treatment also reduced the in vitro IL-4 production from CD3-stimulated liver T cells. The systemic deficiency in the Th1 response to bacterial stimulation thus leads to a Th2-dominant state and may induce an abnormal cellular immune response in the skin accompanied with an overproduction of IgE and a susceptibility to dermatitis in NC mice.     

Depressed IL-12-mediated signal transduction in T cells from patients with Sézary syndrome is associated with the absence of IL-12 receptor beta 2 mRNA and highly reduced levels of STAT4.

Sézary syndrome (SS) is the leukemic phase of cutaneous T cell lymphoma characterized by the proliferation of clonally derived CD4+ T cells that release cytokines of the Th2 T cell phenotype (IL-4, IL-5, IL-10), whereas Th1 T cell cytokines (IL-2, IFN-gamma) are markedly depressed as is expression of IL-12, a pivotal cytokine for Th1 cell differentiation. Normal Th1 cells express both the beta 1 and beta 2 chains of the IL-12 receptor (IL-12R) and tyrosine phosphorylate STAT4 in response to IL-12. Th2 T cells express only the IL-12R beta 1 and thus do not tyrosine phosphorylate STAT4 in response to IL-12. To determine whether SS cells are Th2-like at the level of IL-12 signal transduction, we analyzed RNA from seven patients for the presence of message for the IL-12R beta 1 and beta 2 genes using RNase protection assays and assessed whether IL-12 induced tyrosine-phosphorylation of STAT4 by immunoblotting. In PBL from six of seven SS patients tested, beta 2 message was expressed at low to undetectable levels and its expression could not be stimulated by either IFN-alpha or IFN- gamma, which stimulated beta 2 expression in control PBL. The absence of beta 2 expression is further supportive evidence for the Th2 lineage of SS cells. However, unlike normal Th2 cells, SS cells also showed severely reduced levels of STAT4, suggesting that they have a depressed response to any inducer of the STAT4 signal transduction pathway, including IFN-alpha. This is the first observation linking STAT4 gene expression with a human disease and suggests that dysregulation of STAT4 expression may be significant to the development and/or progression of SS.     

Engineered recombinant ovomucoid third domain can modulate allergenic response in Balb/c mice model

IgE-mediated allergic reactions to egg white are a serious health problem and ovomucoid being the dominant egg white allergen has been on focus in the past decade. Engineered hypoallergens with reduced reactivity for IgE antibodies are being examined to modulate the allergic response and develop prophylactic allergen vaccines. In this study, we evaluated the immunomodulatory effect of a genetic variant of the third domain of ovomucoid (GMFA) which showed reduced IgE binding with egg allergic patient's sera in comparison to the native form of the third domain of ovomucoid (DIII) in a murine model system. Balb/c mice were injected intraperitoneally with DIII and GMFA antigens. Allergen-specific serum IgG, IgG1, IgG2a, and IgE responses were evaluated using enzyme-linked immunosorbent assay. Splenocyte cytokine levels in the medium of the cultured cells were examined by ELISA and levels of IL-4, INF-gamma, and IL-12 (p70) cytokines were quantified. Neutralization with anti-IL-12 monoclonal antibody was assayed and cytokine levels with respect to GMFA mutant antigen stimulation were measured. GMFA mutant form was found to have significantly reduced levels of specific IgE when compared to the DIII suggesting a mutation-induced abrogation of the IgE binding epitope in mice. The increase in IgG2a levels in GMFA together with the decline of IgE and IgG1 points to a shift from a Th2 response to a Th1 dominated response. The cytokine profile showed a modulation of anti-allergic Th1 phenotype in GMFA from a proallergic Th2 response observed with DIII. Low levels of IL-4 and increased levels of INF-gamma and IL-12 were observed and anti-IL-12 monoclonal antibody restored the levels of IL-4 and suppressed levels of INF-gamma and IL-12 in the GMFA sensitized group. These results indicate that GMFA has a marked suppressive effect on the allergic response of ovomucoid and caused a shift towards a Th1 pathway, thereby modulating the Th1/Th2 cytokine balance and could be used as a potential hypoallergenic candidate for allergen-immunotherapy in the treatment of egg white allergy.     

Association of IL-10 receptor 2 (IL10RB) SNP with systemic sclerosis

Interleukin-10 (IL-10) signaling has been suggested to play a role in systemic sclerosis (SSc). IL10RB codes for IL-10 receptor 2 (IL-10R2), a component shared in receptor complexes for IL-10, IL-22, IL-26 and interferon (IFN)-λ. In this study, we examined association of IL10RB polymorphism with susceptibility to SSc. Genotype A/A at rs2834167 (47K/K) was significantly increased in diffuse cutaneous SSc (dcSSc) (41.3% in dcSSc, 20.9% in controls, P = 0.0018, odds ratio = 2.67). A SNP in the 5′ flanking region of IL10RB, rs999788, also showed association with dcSSc; however, this association was shown to be secondarily caused by linkage disequilibrium with rs2834167. Significant association was not observed in limited cutaneous SSc (lcSSc). Presence of anti-topoisomerase I antibody was also associated with rs2834167A/A genotype (P = 0.0019). Serum IL-10 level was significantly associated with the number of rs2834167A allele (P = 0.007). These findings suggested that signaling through IL-10R2 may play a causative role in dcSSc.