Thiols decrease cytokine levels and down-regulate the expression of CD30 on human allergen-specific T helper (Th) 0 and Th2 cells

The thiol antioxidant N-acetyl- L-cysteine (NAC), known as a precursor of glutathione (GSH), is used in AIDS treatment trials, as a chemoprotectant in cancer chemotherapy and in treatment of chronic bronchitis. In vitro, GSH and NAC are known to enhance T cell proliferation, production of IL-2 and up-regulation of the IL-2 receptor. The 120-kD CD30 surface antigen belongs to the tumour necrosis factor (TNF) receptor superfamily. It is expressed by activated T helper (Th) cells and its expression is sustained in Th2 cells. We have analysed the effect of GSH and NAC on the cytokine profile and CD30 expression on human allergen-specific T cell clones (TCC). TCC were stimulated with anti-CD3 antibodies in the presence of different concentrations of GSH and NAC. Both thiols caused a dose dependent down-regulation of IL-4, IL-5 and IFN-gamma levels in Th0 and Th2 clones, with the most pronounced decrease of IL-4. Furthermore, they down-regulated the surface expression of CD30, and the levels of soluble CD30 (sCD30) in the culture supernatants were decreased. In contrast, the surface expression of CD28 or CD40 ligand (CD40L) was not significantly changed after treatment with 20 m M NAC. These results indicate that GSH and NAC favour a Th1 response by a preferential down-regulation of IL-4. In addition, the expression of CD30 was down regulated by GSH and NAC, suggesting that CD30 expression is dependent on IL-4, or modified by NAC. In the likely event that CD30 and its soluble counterpart prove to contribute to the pathogenesis in Th2 related diseases such as allergy, NAC may be considered as a future therapeutic agent in the treatment of these diseases.     

Ectopic expression of activated Stat6 induces the expression of Th2-specific cytokines and transcription factors in developing Th1 cells

Stat6 is critical for IL-4-mediated Th2 cell development, but its molecular mechanism remains unclear. Here we constructed Stat6:ER, a Stat6-estrogen receptor fusion protein that can be activated by 4-hydroxy-tamoxifen, independently of IL-4 and endogenous Stat6. Retrovirus-mediated introduction of Stat6:ER into developing Th1 cells induced Th2-specific cytokines and suppressed IFNgamma production in a 4-HT-dependent manner and in the absence of IL-4. It also induced GATA-3 and c-maf expression and downregulated IL-12Rbeta2 chain expression. Its decreased ability to induce the Th2 phenotype with progressing Th1 cell commitment correlated with a decreased induction of GATA-3 and c-maf. This study indicates that Stat6 functions upstream of GATA-3 and c-Maf to induce Th2 development.     

Resistance of activated human Th2 cells to NO-induced apoptosis is mediated by gamma-glutamyltranspeptidase

Activation-induced death of inflammatory cells (AICD) has an important function in immune maintenance. Type 1 Th cells are known to be more susceptible to AICD than Th2 cells. In the current study we examined whether NO-induced apoptosis also preferentially eliminates Th1 cells over Th2 cells. Naive human Th lymphocytes (CD4(+)CD45RO(-)) were activated in vitro for 1 week in the presence of IL-12 plus anti-IL-4 or IL-4 plus anti-IL-12 to generate Th1- and Th2-polarized cultures respectively. Cultures were exposed to the NO donors Spermine-nonoate (Sper) and DPTA-nonoate to study NO-induced apoptosis. We found that NO preferentially induced apoptosis in Th1-polarized cells as demonstrated by Annexin staining in the presence of 10 microM Sper (70 +/- 16 versus 23 +/- 4.4% in Th2 cells P: < 0.01) and by DioC6 staining (38 +/- 10 versus 11 +/- 5% in Th2 cells, P: < 0.01). The mechanism of NO-induced apoptosis in Th1/Th2-polarized cells was distinct from AICD and Fas-induced apoptosis. Differential sensitivity between Th1- and Th2-polarized cultures originated at the level of intracellular glutathione (GSH) metabolism. GSH levels were higher in Th2 cells (1.6 +/- 0.2-fold Th1, P: < 0.01). High intracellular GSH in Th2-polarized cells did not account for reduced susceptibility to NO per se, since the inhibition of gamma-glutamyltrans-peptidase (gamma-GT), which is involved in GSH import, sensitized Th2 cells to NO-induced apoptosis without GSH depletion. Therefore, higher activity of gamma-GT in Th2 cells (2.1 +/- 0.4-fold Th1, P: < 0.001) specifically protects Th2 cells against NO-induced apoptosis. Preferential NO-induced elimination of human Th1 cells at sites of inflammation may thus select Th2 cells and contribute to immune deviation.     

Role of cysteinyl leukotrienes in human allergen-specific Th2 responses induced by granulocyte macrophage-colony stimulating factor

Background:  The pro-inflammatory cytokine, granulocyte macrophage-colony stimulating factor (GM-CSF), which is elevated in the lungs of atopic asthmatic patients, has been shown to enhance major histocompatibility class II expression of alveolar macrophages (AM). We hypothesized that exposure of AM and monocytes from atopic asthmatic patients to GM-CSF would enhance their antigen presenting function, and investigated putative mechanisms for this effect.
Methods:  Alveolar macrophages were purified from bronchoalveolar lavage by plastic adherence. Monocytes and CD4⁺ T cells were purified from peripheral blood by magnetic bead separation. Antigen-presenting cell (APC) were pretreated with GM-CSF, pulsed with allergen and cocultured with autologous T cells. T-cell proliferation was determined by tritiated thymidine incorporation and cytokine production by enzyme-linked immunosorbent assay.
Results:  Exposure of allergen-pulsed AM and peripheral blood monocytes to GM-CSF significantly increased allergen-specific T-cell proliferation and T helper 2 (Th2) cytokine production. The enhanced response was dependent on costimulation by CD86, but not CD80. Inhibition of the 5-lipoxygenase pathway abrogated GM-CSF-mediated upregulation by monocytes of allergen-specific interleukin-5 (IL-5) and IL-13 cytokine production. Blocking of the cysteinyl leukotriene receptor 1 (cysLT₁) receptor by a specific receptor antagonist inhibited allergen-specific IL-5 production in response to GM-CSF pretreatment.
Conclusion:  Exposure to GM-CSF enhanced the capacity of human APC from atopic asthmatic patients to induce allergen-specific Th2 responses by a mechanism involving cysLT. Novel immunotherapies, targeting production of GM-CSF or its actions on APC have the potential, therefore, to prove beneficial in treatment of patients with inflammatory airway disease.     

Distribution of Th1, Th2, and Th0 and the Th1/Th2 cell ratios in human peripheral and endometrial T cells

PROBLEM: To examine whether normal pregnancy involves type 2 T-helper (Th2) immune condition or not. METHOD OF STUDY: We measured the percentage of Th0, Th1, and Th2 and the Th1/Th2 cell ratios of human peripheral blood and endometrial T cells using flow cytometry, which can analyze both the surface marker CD3, and intracellular cytokines, interleukin 4 (IL-4) and interferon gamma (IFNgamma). RESULTS: No significant differences were found in the percentages of Th1, Th2, and Th0 and the Th1/Th2 cell ratios in the peripheral blood T cells of nonpregnant women and women in early pregnancy. On the other hand, the percentage of Th1 cells was highest during the proliferative phase of the endometrium, followed by the secretory phase and early pregnancy decidua. The percentage of Th2 cells was highest in early pregnancy decidua and lowest during the proliferative phase of the endometrium. The Th1/Th2 ratio was 147.48+/-96.68 during the proliferative phase of the endometrium, 37.74+/-21.33 during the secretory phase, and 1.31+/-0.48 in the early pregnancy decidua. CONCLUSIONS: These data indicate that Th1 cells predominate in the nonpregnant endometrium, especially during the proliferative phase, while Th2 cells predominate in early pregnancy decidua.     

Assessment of chemokine receptor expression by human Th1 and Th2 cells in vitro and in vivo.

The preferential association of some chemokine receptors with human Th1 or Th2 cells has recently been reported. In this study, the expression of CCR3, CCR5, CXCR3, and CXCR4 were analyzed by flow cytometry in three distinct in vitro models of Th1/Th2 polarization, activated naive and memory T cells, and T-cell clones, in which the intracellular synthesis of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) and the surface expression of CD30 and LAG-3 were also assessed. Moreover, by using immunohistochemistry the in vivo expression of CCR3, CCR5, CXCR3, and CXCR4 was examined in the gut of patients suffering from Crohn's disease, a Th1-dominated disorder, and in the skin of patients suffering from systemic sclerosis, a Th2-dominated disorder. CCR5 and LAG-3 exhibited the same pathway of Th1 association, whereas CXCR3 did not discriminate between Th1- and Th2-dominated responses. On the other hand, CCR3 was found only occasionally in a small proportion of allergen-specific memory T cells with Th2/ThO profile of cytokine production in vitro. However, it was neither seen in Th2-polarized activated naive T cells nor in established Th2 clones and could be detected in vivo only on non-T cells. Finally, whereas CXCR4 expression was not limited to Th2 cells in vivo, it was markedly up-regulated by IL-4 and down-regulated by IFN-gamma in vitro. Thus, the results of this study confirm the existence of flexible programs of chemokine receptor expression during the development of Th1 and Th2 cells. However, caution is advised in interpreting these receptors as surrogate markers of a given type of effector response.     

Langerin+ dendritic cells are responsible for LPS-induced reactivation of allergen-specific Th2 responses in postasthmatic mice

Allergic asthma is a T cell-dependent inflammatory lung disease that results from complex interactions between genetic predisposition and environmental factors, including exposure to lipopolysaccharide (LPS). In this study, we have shown that airway LPS exposure was sufficient to induce airway hyperreactivity (AHR) and eosinophil recruitment in mice that had previously experienced an acute episode of allergic asthma. LPS-induced disease reactivation depended on the activation of allergen-specific CD4(+) T cells by a subset of lung langerin(+) dendritic cells (DCs) that retained the allergen. Upon LPS exposure, migration of langerin(+) DCs from lungs to draining lymph nodes increased and LPS-exposed langerin(+) DCs instructed CD4(+) T cells toward a T helper (Th) 2 response. Selective depletion of langerin(+) DCs prevented LPS-induced eosinophil recruitment and T-cell activation, further demonstrating a critical role for langerin(+) DCs in disease reactivation. This finding provides a possible explanation for the subclinical worsening of asthmatics following exposure to low-dose LPS.     

Chromatin remodeling at the Th2 cytokine gene loci in human type 2 helper T cells

The differentiation of mouse na?ve CD4 T cells into type 2 helper (Th2) cells is accompanied by chromatin remodeling at the nucleosomes associated with the IL-4, IL-13 and IL-5 genes. However, little is known about how chromatin remodeling of these Th2 cytokine gene loci occurs in human Th2 cells. We herein established an in vitro culture system in which both Th1 and Th2 cells are efficiently differentiated from human peripheral blood na?ve CD4 T cells. This system allowed us to investigate the chromatin status at the Th2 cytokine gene loci and the IFNgamma locus in human Th2 and Th1 cells, respectively. In typical individuals, the chromatin remodeling indicated by the induction of hyper-acetylation of histone H3 lysine 9 and hyper-methylation of histone H3 lysine 4 was induced at the whole Th2 cytokine gene loci in developing Th2 cells. We more precisely assessed the methylation status of histone H3 lysine 4 at the Th2 cytokine gene loci (IL-5 exon 3, IL-5 promoter, IL-5/RAD50 intergenic region, RAD50 promoter, CGRE, CNS1, IL-13 promoter, IL-4 promoter, and VA enhancer regions) and the IFNgamma locus in developing Th1 and Th2 cells prepared from 20 healthy volunteers. Th2-cell specific chromatin remodeling was induced at most of the Th2 cytokine gene loci. In parallel with the induction of chromatin remodeling, GATA3 mRNA was preferentially expressed in developing Th2 cells, whereas T-bet, HLX and ROG mRNA was selectively expressed in developing Th1 cells.     

Modulation of chemokine receptor expression and chemotactic responsiveness during differentiation of human naive T cells into Th1 or Th2 cells

Chemokines and their receptors direct movements and encounters of lymphocytes and professional APC into specific microenvironments of lymphoid tissues. Chemokine receptors such as CCR7, CXCR5 and CCR4 that are differentially expressed and modulated in distinct subsets of T cells contribute to establish functionally and spatially segregated microenvironments within secondary lymphoid tissues where T cell activation and differentiation occur. Here, we have explored the modulation of CCR7, CCR4, CCR8 and CXCR5 expression and chemotactic responsiveness to their ligands during commitment of human naive T cells along the Th1 or Th2 differentiation pathway in vitro. Our results document that activation of human naive T cells and differentiation in Th1 or Th2 cells result in progressive down-modulation of CCR7 expression and CCL19 responsiveness. By contrast, expression of CCR4 and responsiveness to CCL22 is rapidly induced at the early stages of both Th1/Th2 cell development. However, while CCR4 expression is further up-regulated upon differentiation into Th2 cells, it is lost on fully differentiated Th1 cells. CCR8 is detected at later time points than CCR4 and exclusively on differentiated Th2 cells as revealed by analysis of mRNA expression and responsiveness to CCL1. Expression of CXCR5 is transiently induced at the early stages of Th cell differentiation, but with distinct kinetics in developing Th1 and Th2 cells. Analysis of human tonsillar CD4(+) T cells reveals a consistent pattern of chemotactic responsiveness and chemokine receptor expression in distinct transitional stages of human T cell activation and differentiation in vivo.     

Genomic-scale analysis of gene expression in resting and activated T cells

Recent advances in gene array technology and isolation of lymphocytes now allow comprehensive analysis of gene expression in many different types of T cells. So far only a few sets of results have been published. However it is already clear that these analyses provide accurate measurements of gene expression in T cells. This technology offers the first opportunity to examine global and subtle changes in gene expression in response to specific stimuli.     

Analysis of allelic differential expression in human white blood cells

Allelic variation of gene expression is common in humans, and is of interest because of its potential contribution to variation in heritable traits. To identify human genes with allelic expression differences, we genotype DNA and examine mRNA isolated from the white blood cells of 12 unrelated individuals using oligonucleotide arrays containing 8406 exonic SNPs. Of the exonic SNPs, 1983, located in 1389 genes, are both expressed in the white blood cells and heterozygous in at least one of the 12 individuals, and thus can be examined for differential allelic expression. Of the 1389 genes, 731 (53%) show allele expression differences in at least one individual. To gain insight into the regulatory mechanisms governing allelic expression differences, we analyze a set of 60 genes containing exonic SNPs that are heterozygous in three or more samples, and for which all heterozygotes display differential expression. We find three patterns of allelic expression, suggesting different underlying regulatory mechanisms. Exonic SNPs in three of the 60 genes are monoallelically expressed in the human white blood cells, and when examined in families show expression of only the maternal copy, consistent with regulation by imprinting. Approximately one-third of the genes have the same allele expressed more highly in all heterozygotes, suggesting that their regulation is predominantly influenced by cis-elements in strong linkage disequilibrium with the assayed exonic SNP. The remaining two-thirds of the genes have different alleles expressed more highly in different heterozygotes, suggesting that their expression differences are influenced by factors not in strong linkage disequilibrium with the assayed exonic SNP.