Regulation of interleukin 2 gene expression: discrepancy between enhancer activity and endogenous gene expression

A 630-bp fragment of the human interleukin 2 (IL 2) promoter (+51 to -584) permitted expression of a reporter gene transfected into a mouse T cell lymphoma line (EL4) upon induction with phorbol ester. In contrast, when a human T cell lymphoma (Jurkat) was transfected with the same plasmid and subsequently induced with phorbol ester together with phytohemagglutinin, a very low expression of the transfected gene was observed. The endogenous IL 2 gene was equally well expressed in both cell lines upon induction. Thus, the expression of the endogenous and the transfected gene did not correlate in Jurkat cells. An SV 40 enhancer element cloned 5' of the IL 2 promoter did not result in constitutive expression after transfection to either cell line, rather the IL 2 promoter retained the need for induction. The addition of an enhancer element increased the induced expression of the transfected gene in Jurkat cells into proximity of that observed in induced EL4 cells, indicating that additional sequence elements not contained in the 630-bp fragment are needed for proper expression of the IL 2 gene in Jurkat cells.     

脂氧素对Jurkat T细胞增殖和白细胞介素-2/白细胞介素-2受体表达的影响

目的：研究脂氧素对Jurkat T细胞增殖和白细胞介素-2表达的影响。方法:体外培养Jurkat T细胞，anti-CD3（2 mg/L）和anti-CD28（2 mg/L）单抗刺激Jurkat细胞活化，加入不同浓度脂氧素（0.1 nmol/L-100 nmol/L）共同孵育24h后，加入［3H］-TdR继续孵育6 h，液闪仪测放射性活度；或收集培养上清，ELISA测IL-2水平，收集细胞，流式细胞仪检测细胞表面IL-2受体α亚单位CD25表达，PI染色后经流式细胞仪进行细胞周期分析。结果:脂氧素剂量依赖性抑制anti-CD3和anti-CD28活化的Jurkat T细胞增殖（P<0.05）；细胞周期分析发现脂氧素处理组S期细胞比例减少；脂氧素显著降低培养上清中IL-2 含量（P<0.05）但对CD25表达无明显影响（P>0.05）。结论:脂氧素能抑制活化Jurkat T细胞增殖和白细胞介素-2表达；脂氧素可通过影响T淋巴细胞的活化增殖进而发挥免疫负性调节作用。     

A bidirectional regulatory network involving IL 2 and IL 4 in the alternative CD2 pathway of T cell activation

In this study we examined the effect of interleukin 4 (IL 4) on T cell activation and proliferation via the alternative CD2 pathway. To this end highly purified human resting T cells were cultured with a stimulating pair of anti-CD2 monoclonal antibodies in the absence of accessory signals from monocytes. Addition of either recombinant (r)IL 2 or rIL 4 resulted in proliferation of the anti-CD2-stimulated T cells. The growth-promoting effect of rIL 4 on preactivated. T cells was shown to be partly a direct effect. rIL 4 also induced IL 2 production and, as a consequence, the effect of rIL 4 on T cell growth was enhanced by endogenously produced IL 2. Moreover, rIL 4 acted in synergy with exogenously added rIL 2 in promoting growth of anti-CD2-stimulated T cells. The synergistic effect of IL 2 and IL 4 could be explained by IL 2-induced up-regulation of IL 4 responsiveness. In contrast, preincubation with rIL 4 did not enhance IL 2 responsiveness and rIL 2 but not rIL 4 up-regulated IL 2R expression on anti-CD2-stimulated T cells. Finally we could demonstrate that monocyte-produced cytokines (IL 1 and IL 6) enhance the proliferative response to rIL 4 of anti-CD2-stimulated T cells. It can be concluded that IL 4 can act as a paracrine growth factor for T cells activated in the alternative CD2 pathway, and that it acts synergistically with IL 2, IL 1 and IL 6. Moreover, IL 4 is a helper signal for IL 2 production. Thus, IL 2 and IL 4 are involved in a bidirectional regulatory network, with IL 4 as an inducer of IL 2 production, and IL 2 as an enhancer of IL 4 responsiveness.     

生长激素调节T淋巴细胞的功能

目的：探讨生长激素（ＧＨ）对Ｔ淋巴细胞功能的影响。方法：构建ＧＨ ｃＤＮＡ表达质粒ＰｃＤＮＡ－ＧＨ ｃＤＮＡ，然后将其转染入Ｔ淋巴细胞系－Ｊｕｒｋａｔ细胞中，观察过度表达的内源ＧＨ和加入外源基因重组人ＧＨ对Ｔ淋巴细胞功能的影响。结果：过量表达的内源ＧＨ使Ｊｕｒｋａｔ细胞表达ＩＬ－２Ｒ和分泌ＩＬ－２，ＩＦＮ－γ分别增加１．４０倍，１．６０倍和１．９８倍，而在培养液中加入抗ＧＨ血清后，ＧＨ的这种作用消     

Lineage-specific adjacent IFNG and IL26 genes share a common distal enhancer element

Certain groups of physically linked genes remain linked over long periods of evolutionary time. The general view is that such evolutionary conservation confers 'fitness' to the species. Why gene order confers 'fitness' to the species is incompletely understood. For example, linkage of IL26 and IFNG is preserved over evolutionary time yet Th17 lineages express IL26 and Th1 lineages express IFNG. We considered the hypothesis that distal enhancer elements may be shared between adjacent genes, which would require linkage be maintained in evolution. We test this hypothesis using a bacterial artificial chromosome transgenic model with deletions of specific conserved non-coding sequences. We identify one enhancer element uniquely required for IL26 expression but not for IFNG expression. We identify a second enhancer element positioned between IL26 and IFNG required for both IL26 and IFNG expression. One function of this enhancer is to facilitate recruitment of RNA polymerase II to promoters of both genes. Thus, sharing of distal enhancers between adjacent genes may contribute to evolutionary preservation of gene order.     

Expression of nonmuscle cofilin-1 and steroid responsiveness in severe asthma

BACKGROUND: Glucocorticoids are the mainstay of asthma therapy; however, a proportion of patients with asthma has a severe form of the disease that fails to respond to therapy. Understanding the molecular mechanisms behind glucocorticoid-insensitive asthma is therefore of clinical importance. Evidence in glucocorticoid-unresponsive Henrietta Lack (HeLa) cells indicated that cofilin-1 could act as an inhibitor of glucocorticoid function. OBJECTIVE: To determine whether cofilin-1 expression is abnormally expressed in cells from patients with severe glucocorticoid-insensitive asthma and examine the effect of cofilin-1 overexpression on glucocorticoid function. METHODS: Peripheral blood CD4(+) T cells were purified from 16 subjects with severe glucocorticoid-insensitive asthma and 16 subjects with mild glucocorticoid-sensitive asthma, and cofilin-1 expression was determined by quantitative real-time RT-PCR and Western blotting. The effect of dexamethasone on cofilin-1 expression was determined in Jurkat T cells, and the effect of cofilin-1 overexpression on anti-CD3/CD28-stimulated IL-2 release was measured. RESULTS: Peripheral blood CD4(+) T cells from subjects with severe glucocorticoid-insensitive asthma are less responsive to dexamethasone than cells from subjects with mild glucocorticoid-sensitive asthma. Cells from these patients express significantly (P < .05) higher levels of cofilin-1 than cells from subjects with mild asthma. Dexamethasone did not affect cofilin-1 expression in Jurkat T cells. Functionally, dexamethasone suppression of anti-CD3/CD28-stimulated IL-2 was attenuated in Jurkat cells overexpressing cofilin-1. CONCLUSION: These results suggest that increased cofilin-1 expression may be important in the regulation of glucocorticoid sensitivity in peripheral blood lymphocytes of patients with severe treatment-insensitive asthma. CLINICAL IMPLICATIONS: Understanding the mechanisms of enhanced cofilin-1 expression may lead to the development of new therapies for severe treatment-insensitive asthma.     

Proliferative response of human CD4+ T lymphocytes stimulated by the lectin jacalin

The Galβ(1–3)GalNAc-binding lectin jacalin is known to specifically induce the proliferation of human CD4⁺ T lymphocytes in the presence of autologous monocytes and to interact with the CD4 molecule and block HIV-1 infection of CD4⁺ cells. We further show that jacalin-induced proliferation is characterized by an unusual pattern of T cell activation and cytokine production by human peripheral blood mononuclear cells (PBMC). A cognate interaction between T cells and monocytes was critical for jacalin-induced proliferation, and human recombinant interleukin (IL)-1 and IL-6 did not replace the co-stimulatory activity of monocytes. Blocking studies using monoclonal antibodies (mAb) point out the possible importance of two molecular pathways of interaction, the CD2/LFA-3 and LFA-1/ICAM-1 pathways. One out of two anti-CD4 mAb abolished jacalin responsiveness. Jacalin induced interferon-γ and high IL-6 secretion, mostly by monocytes, and no detectable IL-2 synthesis or secretion by PBMC. In contrast, jacalin-stimulated Jurkat T cells secreted IL-2. CD3^? Jurkat cell variants failed to secrete IL-2, suggesting the involvement of the T cell receptor/CD3 complex pathway in jacalin signaling. IL-2 secretion by CD4^? Jurkat variant cells was delayed and lowered. In addition to CD4, jacalin interacts with the CD5 molecule. Jacalin-CD4 interaction and the proliferation of PBMC, as well as IL-2 secretion by Jurkat cells were inhibited by specific jacalin-competitive sugars.     

Polymorphism of age-related changes in interleukin (IL) production: differential changes of T helper subpopulations, synthesizing IL 2, IL 3 and IL 4

Inbred mice were examined for strain differences in age-associated changes in the capacity to synthesize interleukin (IL), i.e. IL 2, IL 3 and IL 4 after stimulation with phorbol myristate acetate (PMA) and calcium ionophore (A23187). Production of IL 2 remains constant in A/J, DBA/1 and DBA/2 mice and decreases with age in one of the strains examined (C57BL/6J). Strain differences in age-associated change of synthesis did not show the relation between youthful synthetic capacity and magnitude of later decrease ("economic correction") which is observed in several other systems. This difference between different types of polymorphisms is attributed to an age-associated defect in intrinsic capacity to synthesize IL 2 which may occur in only one of the tested strains, C57BL/6J. In contrast to IL 2 production, the quantities of IL 3 and IL 4 increase progressively, with advancing age, in mice of the three strains tested. T cells from old mice contain a greater frequency of cells producing IL 3, than do those of young mice. In addition, synthesis of IL 3 is induced at a relatively lower concentration of PMA in cells from old animals and this may be a consequence of different signal requirements of the two subsets of the T helper cells, but also a change in intrinsic properties of these cells. Since IL 3 and IL 4 production, but not IL 2 production, increases with age, it is reasonable to conclude that this reflects an expansion of a T helper cell population which secretes IL 3 and IL 4, but not IL 2, presumably TH2.     

Evaluation of IL17A expression and of IL17A,IL17F and IL23R gene polymorphisms in Brazilian individuals with periodontitis

The IL23/Th17 axis plays an important role in the pathogenesis of cell-mediated tissue damage caused either by autoimmunity or immune responses against bacterial infection. Single nucleotide polymorphisms in the IL17A, IL17F and IL23R genes have been associated with several inflammatory diseases. However, these polymorphisms have not yet been studied in periodontitis. The aim of present study was to evaluate the expression of IL17A and occurrence of the IL17A (rs2275913), IL17F (rs763780) and IL23R (rs11209026) gene polymorphisms in different clinical forms or severity of periodontitis in a sample of Brazilian individuals. Peripheral blood was obtained from 30 non-smoker individuals and analyzed by flow cytometry to determine IL-17 expression. Genomic DNA was obtained from oral swabs in 180 individuals and analyzed by Real-time PCR. The study group was composed by individuals without periodontitis (control), with aggressive periodontitis (AP) and with chronic periodontitis (CP). Higher frequency of IL17A+CD4+ T cells was observed in control group. The A+ genotype from IL17A (rs2275913) was associated with lack of disease. No association was found considering the IL17F and IL23R polymorphisms. Our data suggest that IL17A and the presence of IL17A (rs2275913) A allele are associated with the absence of periodontal disease.