Ralph C. Budd

Caspase-8 and c-FLIPL Associate in Lipid Rafts with NF-κB Adaptors during T Cell Activation

Humans and mice lacking functional caspase-8 in T cells manifest a profound immunodeficiency syndrome due to defective T cell antigen receptor (TCR)-induced NF-κB signaling and proliferation. It is unknown how caspase-8 is activated following T cell stimulation, and what is the caspase-8 substrate(s) that is necessary to initiate T cell cycling. We observe that following TCR ligation, a small portion of total cellular caspase-8 and c-FLIPL rapidly migrate to lipid rafts where they associate in an active caspase complex. Activation of caspase-8 in lipid rafts is followed by rapid cleavage of c-FLIPL at a known caspase-8 cleavage site. The active caspase·c-FLIP complex forms in the absence of Fas (CD95/APO1) and associates with the NF-κB signaling molecules RIP1, TRAF2, and TRAF6, as well as upstream NF-κB regulators PKCθ, CARMA1, Bcl-10, and MALT1, which connect to the TCR. The lack of caspase-8 results in the absence of MALT1 and Bcl-10 in the active caspase complex. Consistent with this observation, inhibition of caspase activity attenuates NF-κB activation. The current findings define a link among TCR, caspases, and the NF-κB pathway that occurs in a sequestered lipid raft environment in T cells.

Redox-Based Regulation of Apoptosis: S-Glutathionylation As a Regulatory Mechanism to Control Cell Death

SIGNIFICANCE Redox-based signaling governs a number of important pathways in tissue homeostasis. Consequently, deregulation of redox-controlled processes has been linked to a number of human diseases. Among the biological processes regulated by redox signaling, apoptosis or programmed cell death is a highly conserved process important for tissue homeostasis. Apoptosis can be triggered by a wide variety of stimuli, including death receptor ligands, environmental agents, and cytotoxic drugs. Apoptosis has also been implicated in the etiology of many human diseases. RECENT ADVANCES Recent discoveries demonstrate that redox-based changes are required for efficient activation of apoptosis. Among these redox changes, alterations in the abundant thiol, glutathione (GSH), and the oxidative post-translational modification, protein S-glutathionylation (PSSG) have come to the forefront as critical regulators of apoptosis. CRITICAL ISSUES Although redox-based changes have been documented in apoptosis and disease pathogenesis, the mechanistic details, whereby redox perturbations intersect with pathogenic processes, remain obscure. FUTURE DIRECTIONS Further research will be needed to understand the context in which of the members of the death receptor pathways undergo ligand dependent oxidative modifications. Additional investigation into the interplay between oxidative modifications, redox enzymes, and apoptosis pathway members are also critically needed to improve our understanding how redox-based control is achieved. Such analyses will be important in understanding the diverse chronic diseases. In this review we will discuss the emerging paradigms in our current understanding of redox-based regulation of apoptosis with an emphasis on S-glutathionylation of proteins and the enzymes involved in this important post-translational modification.

Cellular FLIP long form augments caspase activity and death of T cells through heterodimerization with and activation of caspase-8

Caspase activity is required not only for the death of T cells, but also for their activation. A delicate balance of caspase activity is thus required during T cell activation at a level that will not drive cell death. How caspase activity is initiated and regulated during T cell activation is not known. One logical candidate for this process is cellular FLIP long form (c-FLIPL), because it can block caspase-8 recruitment after Fas (CD95) ligation as well as directly heterodimerize with and activate caspase-8. The current findings demonstrate that after T cell activation, caspase-8 and c-FLIPL associate in a complex enriched for active caspases. This occurs coincidently with the cleavage of two known caspase-8 substrates, c-FLIPL and receptor interacting protein 1. Caspase activity is higher in wild-type CD8+ than CD4+ effector T cells. Increased expression of c-FLIPL results in augmented caspase activity in resting and effector T cells to levels that provoke cell death, especially of the CD8 subset. c-FLIPL is thus not only an inhibitor of cell death by Fas, it can also act as a principal activator of caspases independently of Fas.

Spatial differences in active caspase-8 defines its role in T-cell activation versus cell death.

Caspase-8, a cysteine-protease, initiates apoptosis when activated by death receptors. Caspase-8 is also essential for initiating T lymphocyte proliferation following T-cell antigen receptor (TCR) signaling. Given these disparate functions of caspase-8, we sought to determine whether this represented only a difference in the magnitude of caspase-8 activation, or different intracellular locations of active caspase-8. We demonstrate by high-resolution multicolor confocal laser scanning microscopy an aggregation of active caspase-8 within membrane lipid rafts in T cells stimulated with anti-CD3. This suggests that following TCR stimulation active caspase-8 physically interacts with lipid raft proteins, possibly to form a signaling platform. In contrast, Fas stimulation of T cells resulted in a much more profound activation of caspase-8 that was exclusively cytosolic. These confocal microscopic findings were confirmed using discontinuous sucrose gradient ultracentrifugation to isolate lipid raft versus cytosolic components. This sequestration model of caspase-8 activation was further supported by the observation that a classic caspase-8 substrate, BID, was not cleaved in CD3-stimulated T cells, but was cleaved after Fas engagement. Our data support a model that the location of active caspase-8 may profoundly influence its functional capacity as a regulator of either cell cycling or cell death.

High Expression of Fas Ligand by Synovial Fluid-Derived γδ T Cells in Lyme Arthritis

γδ T cells accumulate at epithelial barriers and at sites of inflammation in various infectious and autoimmune diseases, yet little is understood about the function of tissue-infiltrating γδ T cells. We observe that γδ T cells of the Vδ1 subset accumulate in synovial fluid of human Lyme arthritis and are intensely cytolytic toward a wide array of target cells. Particularly striking is that the cytolytic activity is highly prolonged, lasting for at least 3 wk after stimulation of the γδ T cells with Borrelia burgdorferi. Cytolysis is largely Fas dependent and results from very high and prolonged expression of surface Fas ligand, which is transcriptionally regulated. This also manifests in a substantial level of self-induced apoptosis of the γδ T cells. In this capacity, certain γδ T cell subsets may serve as cytolytic sentinels at sites of inflammation, and perhaps at epithelial barriers.

Cellular FLIP Long Form-Transgenic Mice Manifest a Th2 Cytokine Bias and Enhanced Allergic Airway Inflammation

Cellular FLIP long form (c-FLIPL) is a caspase-defective homologue of caspase-8 that blocks apoptosis by death receptors. The expression of c-FLIPL in T cells can also augment extracellular signal-regulated kinase phosphorylation after TCR ligation via the association of c-FLIPL with Raf-1. This contributes to the hyperproliferative capacity of T cells from c-FLIPL-transgenic mice. In this study we show that activated CD4+ T cells from c-FLIPL-transgenic mice produce increased amounts of Th2 cytokines and decreased amounts of Th1 cytokines. This correlates with increased serum concentrations of the Th2-dependent IgG1 and IgE. The Th2 bias of c-FLIPL-transgenic CD4+ T cells parallels impaired NF-κB activity and increased levels of GATA-3, which contribute, respectively, to decreased IFN-γ and increased Th2 cytokines. The Th2 bias of c-FLIPL-transgenic mice extends to an enhanced sensitivity to OVA-induced asthma. Taken together, these results show that c-FLIPL can influence cytokine gene expression to promote Th2-driven allergic reaction, in addition to its traditional role of blocking caspase activation induced by death receptors.

IL-15 maintains T-cell survival via S-nitrosylation-mediated inhibition of caspase-3

Caspase activity is critical for both T-cell survival and death. However, little is known regarding what determines caspase activity in cycling T cells. Interleukin (IL)-2 and IL-15 confer very different susceptibilities to T-cell death. We therefore considered that IL-2 and IL-15 differentially regulate caspase activity to influence T-cell survival. We observed that IL-2-cultured primary murine effector T cells manifested elevated levels of caspase-3 activity compared with IL-15-cultured T cells. T cell receptor (TCR) restimulation further increased caspase activity and induced considerable cell death in IL-2-cultured T cells, but provoked only a minimal increase of caspase activity and cell death in IL-15-cultured T cells. IL-2 sensitization to cell death was caspase-3 mediated. Interestingly, increased active caspase-3 levels with IL-2 were independent of active initiator caspase-8 and caspase-9 that were similar with IL-2 and IL-15. Rather, caspase-3 activity was inhibited by posttranslational S-nitrosylation in IL-15-cultured T cells, but not in the presence of IL-2. This paralleled increased reactive nitrogen and oxygen species with IL-15 and reduced glycolysis. Taken together, these data suggest that the metabolic state conferred by IL-15 inhibits T-cell apoptosis in part by maintaining low levels of active caspase-3 via S-nitrosylation.

Fas Ligand Deficiency Impairs Host Inflammatory Response against Infection with the Spirochete Borrelia burgdorferi

ABSTRACT Lyme disease represents a complex response to Borrelia burgdorferi that involves both bacterial factors as well as host responses. This results in an inflammatory reaction at several sites, including the synovial lining of joints. Synovial tissues of inflamed joints contain cells expressing high levels of Fas and Fas ligand (FasL). Although Fas stimulation is typically associated with cell death, it can also transmit stimulatory signals to certain cell types. Among these are dendritic cells and macrophages, which are abundant in inflamed synovium. To better assess the role of FasL in the pathogenesis of Lyme arthritis, we evaluated the response to B. burgdorferi infection in C3H/HeJgld mice that bear a nonfunctional mutation in FasL. Compared to wild-type C3H+/+ mice, C3Hgld mice had a similar bacterial burden and antibody response 2 weeks and 4 weeks following infection, but they manifested a significantly reduced Borrelia-specific cytokine response. In addition, C3Hgld mice developed a greatly reduced incidence and severity of arthritis. The findings document a contribution of FasL to the host inflammatory response to B. burgdorferi.

Expression of T‐Cell Receptors by Functionally Distinct Subsets of Immature Adult Thymocytes

During the intrathymic differentiation of CD4. CD8and CD4CD8 ’ T cells, the genes encoding heterodimeric a/P T-cell receptors (R) undergo rearrangement, expression. and selection.’ Recently. a population of so-called double negative (DN) , that is CD4CD8-, thymocytes has been described, which reconstitute in uiuo all other thymocyte subsets.’ The majority of adult thymocytes, essentially most cortical thymocytes. coexpress both CD4 and CD8. About half of these cells also express CD3-associated a/P T-cell R.’ generally at lower intensity than medullary thymocytes. which al-e the most mature population and presumably seed peripheral lymphoid organs. Nearly all CD4‘ CD8 ’ thymocytes. however, die i r i Accumulating evidence indicates that CD4 and CDX enhance T-cell recognition of MHC class I 1 and class I molecules. and because of this, i t is thought that selective events in the thymus involve CD4 and/or CD8. Recent experiments have demonstrated clearly that negative selection occurs by clonal elimination of self-reactive cells,’-’ although the role of positive selection or “education“ for self-MHC restriction elements is still controversial. Our laboratory has primarily studied the DN thymocyte subsets. Analysis of DN cells by several criteria has indicated that this population is itself quite heterogeneous. l‘ l . ’ i These findings implicate thc existence of a number of subpopulations: however, the ontogenic and functional relationships between these subsets is largely unknown. We will summarize in this paper studies that have identified four major phenotypically distinct subsets differing in cell-cycle status and i r i uirro activation requirements. We have further focused biochemical and molecular studies on two subpopulations that have opposing patterns of T-cell K expression. Both y/6 and a/P C D h s s o c i a t e d T-cell Ks have been recently identified on DN thymocytes.”.” Although a/@ T-cell Rs are now understood to be triggered by MHC molecules generally associated with antigenic peptides.I4 the specificity of y/S T-cell Rs remains unknown. Moreover. the functional role of y/S cells and the relationship between the ylfi and a/@ lineages remain5 unclear.

Phenotypic Identification of Memory Cytolytic T Lymphocytes in a Subset of Lyt‐2+ Cells

Murine peripheral Lyt-2+ T cells could be subdivided according to surface expression of the Pgp-1 glycoprotein into major (71%) Pgp-1- and minor (29%) Pgp-1+ subsets. A striking correlation was observed between Pgp-1 expression and enrichment for antigen-specific memory cytolytic T lymphocyte precursors (CTLp). After immunization with the male minor transplantation antigen H-Y, virtually all the H-Y-specific CTLp were found in the minor Pgp-1+ subset of Lyt-2+ cells. In addition, after alloimmunization the frequency of allospecific CTLp resistant to inhibition by anti-Lyt-2 antibody was markedly enriched within the Pgp-1+ cells, suggesting an enrichment for CTLp bearing high avidity antigen receptors. Taken together, these data suggest that surface Pgp-1 expression is stably acquired at the time of primary antigenic stimulation by virgin T cells. As such, Pgp-1 represents an important marker for identifying a subset of Lyt-2+ T cells with the quantitative and qualitative properties of memory CTLp.