Inhibition of activation of transcription factor AP-1 by CD28 signalling in human T-cells

and activation of the CD28 surface molecule results in enhanced proliferation and interleukin 2 (IL-2) production _ The increase in IL-2 gene expression triggered by CD28 involves a KB-like sequence in the 5 '-regulatory region of the IL-2 promoter, called CD28-responsive element. Stimulation of T-cells by agonistic anti-CD28 antibodies in conjunction with phorbol 12myristate 13-acetate (PMA)- or TcR-derived signals induces the enhanced activation of the transcription factor NF-KB. Here we report that CD28 engagement, however, exerts opposite effects on the transcription factor AP-l. Whereas anti-CD28 together with PMA increased the DNA binding and trans-activation activity ofNF-KB, PMA-induced activation of AP-l was significantly suppressed. The inhibitory effect exerted by anti-CD28 was observed at the level of DNA binding as well as in functional reporter-gene assays. These results suggest that the two transcription factors are independently regulated and may perform different functions during T-cell activation

The EMBO Journal vol.14 no.15 pp.3731-3740, 1995 IL-2 gene expression and NF-lCB activation through

Communicated by P.A.Baeuerle Activation of the CD28 surface receptor provides a major costimulatory signal for T cell activation resulting in enhanced production ofinterleukin-2 (IL-2) and cell proliferation. In primary T lymphocytes we show that CD28 ligation leads to the rapid intracellular formation of reactive oxygen intermediates (ROIs) which are required for CD28-mediated activation of the NF-KB/CD28-responsive complex and IL-2 expression. Delineation of the CD28 signaling cascade was found to involve protein tyrosine kinase activity, followed by the activation of phospholipase A2 and 5-lipoxygenase. Our data suggest that Iipoxygenase metabolites activate ROI formation which then induce IL-2 expression via NF-KB activation. These findings should be useful for therapeutic strategies and the development of immunosuppressants targeting the CD28 costimulatory pathway

Eur. J. Immunol. 1995.25: 159-165 Hydrogen peroxide as a potent activator of T lymphocyte functions 159

Hydrogen peroxide as a potent activator of T lymphocyte functions During inflammatory processes infiltrating cells produce large amounts of reactive oxygen intermediates (ROI). Increasing evidence suggests that ROI besides being cytotoxic may act as important mediators influencing various cellular and immunological processes. In this study, we have investigated the effects of hydrogen peroxide on several aspects of lymphocyte activation. In ESb-L T lymphoma cells, micromolar concentrations of hydrogen peroxide rapidly induced activation of the transcription factor NF-xB, whereas DNAbinding activity of the transcription factor AP-1 was virtually not affected. In addition, hydrogen peroxide induced early gene expression of interleukin-2 (IL-2) and the IL-2 receptor a chain. The stimulation of IL-2 expression was found to be conferred by a xB-like cis-regulatory region within the IL-2 gene promoter. In contrast to these activating effects, addition of hydrogen peroxide was largely inhibitory on cell proliferation which is consistent with a general requirement of thiol compounds for lymphocyte proliferation. However, hydrogen peroxide significantly increased T cell proliferation when applied for a short period under reducing conditions. These data indicate that ROI may act as an important competence signal in T lymphocytes inducing early gene expression as well as cell proliferation.

The EMBO Journal vol.14 no.3 pp.546-554. 1995 HIV-1 Tat potentiates TNF-induced NF-KB activation and cytotoxicity by altering the cellular redox state MichaeIO.Westendorp1,

M.O.Westendorp and V.A.Shatrov contributed equally to this stud

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response