Biological activity of recombinant murine interleukin-6 in interleukin-1 T cell assays

Interleukin-6 (also called B cell stimulatory factor 2, hepatocyte activating factor, interferon-β₂) has been shown to have effects on various lineages of hemopoietic cells. Some of its activities appear to overlap those of interleukin-1. In particular, recombinant murine IL-6 induced proliferation of phytohemagglutinin-activated thymocytes, an assay widely used to detect IL-1. In this report, we compared several features of IL-1 and IL-6 dependent thymocyte proliferation. The results indicate that IL-2 is the major second mediator of both IL-1 and IL-6 dependent proliferation. Finally, we tested whether IL-6 would also have activity in other T cell-based IL-1 assays using the T cell lymphoma LBRM33 1A5 and the T cell clone D10-G4.1. IL-6 had no activity in the latter two assays. These results indicate that IL-1 assays using LBRM33 1A5 and D10-G4.1 selectively detect Il-1, and are more specific assays for the detection of IL-1 in samples that may also contain IL-6.     

Interleukin-7 regulates c-myc expression in murine T cells and thymocytes: a role for tyrosine kinase(s) and calcium mobilization

Interleukin-7 (IL-7) was originally identified as a pre-B cell growth factor whose proliferating activity has been extended to numerous target cells including T lymphocytes. We investigated c-myc mRNA expression, an oncogene associated with proliferation, in the murine T cell line D10 G4.1 and freshly isolated thymocytes since both target cells proliferate in response to IL-7. We find that blockade of the tyrosine kinase pathway by genistein, a potent tyrosine kinase inhibitor, inhibits both IL-7-dependent D10 G4.1 cell proliferation and c-myc mRNA expression which appears to involve de novo mRNA synthesis and to be under the control of short-lived protein repressor(s). We have also examined possible signal transduction pathways which might regulate c-myc mRNA expression in the murine T cell line. IL-7 biological activity is not affected by stimulation of the protein kinase C pathway by phorbol esters. Thus, IL-7 regulates c-myc mRNA expression in a protein kinase C-independent manner and these data are strengthened by protein kinase C depletion which does not modify IL-7 c-myc mRNA responsiveness. In contrast and independent of protein kinase C activation, intracellular calcium mobilization by means of ionomycin reduces IL-7 induction of c-myc mRNA expression and may represent a physiological mechanism whereby IL-7 bioactivity is regulated. The activity of IL-7 on c-myc mRNA expression has been extended to freshly isolated thymocytes and we find a synergistic effect of IL-7 with concanavalin A. Taken together our results illuminate the molecular mechanism of IL-7 c-myc induction in the T lineage by ascribing a role for tyrosine kinase and increase in intracellular calcium in both IL-7 induced gene induction and cell proliferation.     

IL-7及其受体在T淋巴细胞发育中的作用及对运动免疫的意义

运动训练显著影响机体的免疫功能,T淋巴细胞亚群的平衡是维持免疫系统内部环境稳定的一个中心环节.IL-7/IL-7受体(IL-7R)在促进淋巴细胞,尤其是T淋巴细胞产生、分化和发育成熟的过程中发挥重要作用.从了解IL-7/IL-7 R的生物学特性、信号通路及其在T细胞发育、生存、扩增和记忆性T细胞发育中的重要作用,从细胞免疫产生的源头来分析IL-7/IL-7R运动影响免疫功能的机理,可为改善机体免疫功能提供新的思路.     

Alamar Blue-B13细胞株生物法测定IL-5水平

目的 探讨应用Alamar Blue测定细胞因子IL-5的方法。方法 将依赖IL-5的B13细胞加到细胞培养板上,加入系列稀释的待测样品IL-5/CHO细胞培养上清,孵育4d后加入Alamar Blue,继续孵育6h测定每孔A值(A_(570)-A_(600))。结果 随着样品中IL-5浓度的升高,B13细胞增殖程度增加,A值增大。敏感性较生物素ELISA法提高12～15倍。结论 AlamarBlue-B13细胞株生物法是一种测定IL-5的可行的、无放射性污染的、敏感的方法。     

T cell development, ageing and Interleukin-7

Interleukin-7 (IL-7) is a cytokine with a central role in the development and maintenance of the peripheral T cell pool. In the mouse, expression of the IL-7 gene in the thymus has been carefully followed from gestation onwards throughout the lifespan. One of the features of its expression in the thymus is that it changes with time, declining measurably as the animal ages. This reduction is associated with a decrease in thymic size, cellularity and output. Analysis of transgenic animals carrying either IL-7 or IL-7 receptor transgenes reveals that the intrathymic level of IL-7 has a critical effect on the production of T cells, and that this may not be a linear relationship. This is an important consideration for therapy involving treatment of old animals with IL-7 of which there are reports indicating some rejuvenation of the thymus following IL-7 treatment, which is never complete. The thymus does not appear to return to the size and cellularity seen in youth. Several possible scenarios could account for this, including the inability to maintain IL-7 within defined limits in the thymus during the therapy.     

IL-7及其受体与干燥综合征关系的研究进展

IL-7是一种具有多种生物学功能的细胞因子,通过影响T、B淋巴细胞的成熟、分化及其他细胞因子的分泌,在炎症的发生发展过程中起到了重要作用.干燥综合征(SS)是一种以外分泌腺体高度淋巴细胞浸润和淋巴瘤高发生率为特征的炎症性自身免疫性疾病.IL-7/IL-7R的高表达与SS的发病机制及病情进展关系密切,因此通过封锁IL-7/IL-7R可能会阻止SS患者炎症和组织破坏的发生,可能会为临床治疗SS提供一种新的途径.     

Development of T cell precursor activity in the murine fetal liver

The generation of T cell precursors in the liver of murine embryos was studied. The total number of T cell precursors in the liver was measured in thymic organ cultures by a limiting dilution assay. Sixty T cell precursors were detected in the liver at day 11 of gestation. By day 12 the number of precursors showed a 20-fold increase, half of which could be explained by in situ proliferation as ascertained by a fetal liver organ culture assay. By day 13 a further 2 – 3-fold increase was observed. Whereas the number of total liver cells continued to increase, that of T cell precursors declined in the following days, suggesting a massive exit of these cells after day 13. The capacity to generate a TCRB repertoire in the cells was evaluated by a PCR assay. T cell precursors in day 11 fetal liver developed a TCRB repertoire at day 8 of culture. The cells from days 12 – 15 developed an identically diverse repertoire by day 6, suggesting that day 11 precursors are more immature than those of later days. A mechanism for yielding a single wave of T cell precursors in the fetal liver is discussed with a proposed model.     

Reciprocal expression of co-stimulatory molecules,B7-1 and B7-2, on murine T cells following activation

The co-stimulatory B7 molecules (B7-1 and B7-2) are expressed on professional antigen-presenting cells in mice. In this study, we demonstrate that B7-1 (CD80) and B7-2 (CD86) are also expressed on murine T cells in the absence of major histocompatibility complex class II molecules. The temporal expression of these two molecules on T cells varies with the state of activation where resting T cells express B7-2 but show little or no expression of B7-1. Following activation, B7-2 expression is down-regulated and there is a concomitant increase in the expression of B7-1 on the cell surface which peaks at about 72 h. Thus these two co-stimulatory molecules are reciprocally expressed on the T cell surface. This pattern of expression of B7-1 and B7-2 on T cells suggests that these two molecules may have different roles in the generation and regulation of immune responses.     

膀胱肿瘤细胞产生的白介素-10对T细胞功能的抑制作用

目的 :了解膀胱肿瘤细胞产生的白介素 10对T细胞功能的抑制作用。方法 :用ELISA法检测膀胱肿瘤细胞株EJ、T2 4和正常移行上皮细胞培养上清中IL 10含量。用MTT法检测加与不加IL 10拮抗剂时膀胱肿瘤细胞株EJ、T2 4和正常移行上皮细胞对共培养的T细胞增殖情况的影响。结果 :EJ和T2 4细胞培养上清中IL 10水平明显高于正常移行上皮和T细胞 (P <0 0 1)。EJ组与IL 10阻断EJ组比较 ,T细胞的增殖反应降低 (P <0 0 5 ) ;IL 10阻断EJ组与IL 10阻断正常移行上皮组、正常移行上皮组、IL 10阻断T细胞组和T细胞组比较 ,T细胞的增殖反应降低 (P <0 0 5 ) ;T2 4组与IL 10阻断T2 4组比较 ,T细胞的增殖反应降低 (P <0 0 5 ) ,IL 10阻断T2 4组与IL 10阻断正常移行上皮组、正常移行上皮组、IL 10阻断T淋巴细胞组和T淋巴细胞组比较T淋巴细胞的增殖反应降低 (P <0 0 5 )。结论 :膀胱肿瘤细胞株EJ、T2 4细胞可通过产生IL 10 ,抑制T细胞增殖能力 ,但可能还存在其它机制。     

Thymic selection and peptide-induced activation of T cell receptor-transgenic CD8 T cells in interleukin-2-deficient mice

The requirement for interleukin-2 (IL-2) in repertoire selection and peripheral activation of CD8 T cells was tested in mice rendered IL-2 deficient by gene targeting and expressing a transgenic T cell receptor (TcR) (F5) specific for influenza nucleoprotein (NP) 366-374 + H-2D^b. Positive selection of the transgenic F5 TcR into the CD8 compartment proceeded normally. Both in vivo and in vitro, the antigenic peptide induced depletion of immature thymocytes and proliferation of mature CD8 T cells regardless of the presence of an intact IL-2 gene. In contrast, cytotoxic T lymphocyte (CTL) activity was only generated by T cells from IL-2⁺ F5 transgenic mice. Exogenous IL-2 was able to fully restore the CTL response of IL-2^?/? responder cells in vitro. Thus, both in vivo and in vitro, clonal expansion of CD8 T cells can proceed in the absence of IL-2, whereas in peptide-immunized F5 transgenic mice, induction of cytotoxic effector function is IL-2 dependent.     

Dysregulation of interleukin-7 receptor may generate loss of cytotoxic T cell response in human immunodeficiency virus type 1 infection

Virus-specific cytotoxic T lymphocytes (CTL) play a crucial role in modulating an immune response against human immunodeficiency type 1 (HIV-1) infection. The generation of effector cytotoxic cells from CTL precursors involves intricate interactions with antigen via T cell receptors (TcR) and soluble cytokines. Interleukin (IL)-7 can affect T cell maturation and differentiation. Here we report on a group of five HIV-1-positive individuals who tested negative for env-and gag-specific CTL activity. When exogenous recombinant human IL-7 was added as a stimulus to the cultures, none (0/5) of the CTL-negative individuals exhibited a CTL response. Individuals that were negative for HIV-1-specific CTL activity were found to lack IL-7 receptor (IL-7R) on CD8⁺ cells with a comparable reduction on CD4⁺ cells. Increased shedding of IL-7R in the culture supernatant was observed. A significant reduction in receptor number was detected by binding of ¹²⁵I-labeled IL-7 and Scatchard analysis. The lack of IL-7R is probably not due to endogenous IL-7, since it was not detectable in the culture supernatants of the patients studied. HIV-1 proteins may cause down-modulation of IL-7R expression, either by producing an insufficient number of molecules or by rapid decay of IL-7R on T cells. These changes may alter the cells' capability to respond to the IL-7 growth signal, resulting in CTL failure and subsequent mishandling of the virus.     

The Serum Factor from Patients with Ulcerative Colitis that Induces T Cell Proliferation in the Mouse Thymus Is Interleukin-7

The disturbance of immune regulatory T cells is related to the pathogenesis of ulcerative colitis. Here we demonstrated and characterized the serum factor from ulcerative colitis patients that induced proliferation of intrathymic T cells. The factor isolated from the patient sera by a combination of gel filtration and anion-exchange chromatography induced proliferation of CD4⁺CD8^– intrathymic T cells in the organ-cultured embryonic mouse thymus. Purification and amino acid sequence analysis of the serum factor demonstrated that the N-terminal 12 sequence was homologous to that of interleukin-7. SDS-PAGE and Western blot confirmed that purified serum factor was interleukin-7. Enzyme immunoassay demonstrated that the serum interleukin-7 concentration was significantly increased in the patients. PCR and Southern blot hybridization demonstrated that interleukin-7 mRNA expression was increased in the thymus tissues from patients but decreased in the colonic mucosa. Since interleukin-7 is a crucial cytokine for proliferation and differentiation of T cells in the thymus, the present study indicates that interleukin-7 may contribute to the disturbance of immune regulatory T cells in ulcerative colitis.     

A transgenic T cell receptor restores thymocyte differentiation in interleukin-7 receptor α chain-deficient mice

Interleukin-7 (IL-7) receptor α chain-deficient (IL-7Rα^-/-) mice have severely depleted lymphocyte populations and thymocyte development is arrested at the double-negative (DN) stage. We show that thymocyte development in these mice can be reconstituted by the introduction of a transgenic T cell receptor (TCR), implying that one function of the IL-7Rα chain is to initiate TCR gene rearrangement. Expression of the recombinase-activating genes RAG1 and RAG2 was greatly reduced in the IL-7Rα^-/- thymuses, and in DN thymocytes from the TCR transgenic IL-7Rα^-/- mice, but was restored in double-positive thymocytes from the TCR transgenic IL-7Rα^-/- mice. These data suggest that the IL-7Rα chain controls RAG expression and initiation of TCRβ chain VDJ rearrangement in DN cells. In contrast, once cells have progressed beyond the DN stage of development the IL-7Rα chain becomes no longer essential for RAG expression.     

T cell sensitivity to HLA class I-mediated apoptosis is dependent on interleukin-2 and interleukin-4

Antibody interaction with a specific epitope of the HLA class I α1 domain triggers apoptosis of activated but not resting T and B cells by a pathway which involves neither Fas ligand nor tumor necrosis factor-α. We have investigated at which stage of activation and proliferation T cells become sensitive to HLA class I-mediated apoptosis, using two monoclonal antibodies (mAb) which recognize the same monomorphic epitope of the HLA class I α1 domain (mAb90, mouse IgG1, and YTH862, rat IgG2b) and can induce apoptosis of phytohemagglutinin (PHA)-activated peripheral blood lymphocytes. Sensitivity to apoptosis develops after the expression of G1 markers (CD69 expression) but it is accelerated by addition of recombinant interleukin-2 (rIL-2). Blocking the IL-2 pathway by cyclosporin A, FK506, rapamycin, anti-IL-2 or CD25 antibodies, prevented the development of sensitivity to apoptosis. Addition of IL-2 and, to a lesser extent, IL-4, reversed the inhibitory effect of cyclosporin A. Conversely, rIL-7 and recombinant interferon-γ restored proliferation of peripheral blood lymphocytes stimulated by PHA in the presence of cyclosporin A but did not restore sensitivity to class I-mediated apoptosis. Finally cells stimulated in the presence of the DNA polymerase inhibitor aphidicolin did not enter into S phase of the cell cycle but secreted IL-2 and underwent apoptosis when exposed to mAb90 or YTH862. Together, the data indicate that sensitivity of peripheral T cells to HLA class I-mediated apoptosis depends on both activation signals and IL-2 or IL-4, but does not require cell proliferation. These data suggest that YTH862 and mAb90 might be used for achieving clonal deletion of antigen-activated peripheral T cells in vivo, provided that the IL-2 pathway is not blocked by other immunosuppressive agents.     

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.     

Functional CD40 ligand expression on T lymphocytes in the absence of T cell receptor engagement: involvement in interleukin-2-induced interleukin-12 and interferon-γ production

Despite the fact that the great majority of T cells at the site of an inflammatory response are not antigen specific, the mechanisms leading to activation and recruitment of these bystander T cells are poorly understood. We previously reported that soluble (s)CD23 potentiated the interleukin (IL)-2-induced interferon (IFN)-γ production by T cells co-cultured with autologous monocytes in the absence of T cell receptor (TCR) engagement. Our present data demonstrate that the IL-2-induced IFN-γ secretion, in the presence but also in the absence of sCD23, is strictly IL-12 dependent, inasmuch as anti-IL-12 antibody abrogated both responses. Most interestingly, anti-CD40 ligand (CD40L) monoclonal antibody significantly inhibited IL-2-induced IL-12 as well as IFN-γ production. These results suggest that CD40L was expressed on T cells in the absence of TCR engagement. Indeed, purified unstimulated T cells readily expressed CD40L. IL-2 and monocytes did not up-regulate CD40L on resting T cells. It is proposed that low levels of CD40L expression on non-antigen stimulated T cells are sufficient to signal through CD40 molecules on accessory cells and to induce IL-12 secretion, which in turn can synergize with IL-2 for the induction of IFN-γ production, thus contributing to the inflammatory process.     

Interferon-γ and interleukin-4 regulate T cell interleukin-12 responsiveness through the differential modulation of high-affinity interleukin-12 receptor expression

Interferon-γ (IFN-γ) and interleukin-4 (IL-4) are mutually antagonistic cytokines that stimulate CD4⁺ T cells to develop into either Th1 or Th2 cells. One feature of Th2 differentiation in mice is the loss of IL-12-induced Jak2 and Stat4 activation, which is accompanied by the inability to produce IFN-γ in response to IL-12. In this report, we show that freshly isolated human T cells activated with phytohemagglutinin (PHA) in the presence of IL-4 exhibit a greatly diminished response to IL-12, whereas the IL-12 response of T cells activated with PHA plus IFN-γ is enhanced. Radiolabeled IL-12 binding studies demonstrate that the impairment of T cell IL-12 responsiveness by IL-4 is associated with the down-regulation of high-affinity IL-12 receptor expression. In contrast, the enhancement of IL-12 responsiveness by IFN-γ is associated with the up-regulation of high-affinity IL-12 receptor expression. Through the use of a newly synthesized neutralizing antibody to the low-affinity IL-12 receptor β subunit (IL-12Rβ), we show that neither IL-4 nor IFN-γ affect the expression of IL-12Rβ, which we determine to be one of at least two low-affinity subunits required for high-affinity IL-12 binding. These findings suggest that IL-4 and IFN-γ exert opposite effects on T cell IL-12 responsiveness by differentially modulating the expression of low-affinity IL-12 receptor subunits that are distinct from IL-12Rβ and required, together with IL-12Rβ, for high-affinity IL-12 binding and IL-12 responsiveness. This provides a basis for understanding the interplay between different cytokines at the level of cytokine receptor expression, and offers insight into one of the mechanisms governing Th1 and Th2 development.