Mark S. Williams

Discrete Generation of Superoxide and Hydrogen Peroxide by T Cell Receptor Stimulation: Selective Regulation of Mitogen-Activated Protein Kinase Activation and Fas Ligand Expression

Receptor-stimulated generation of reactive oxygen species (ROS) has been shown to regulate signal transduction, and previous studies have suggested that T cell receptor (TCR) signals may involve or be sensitive to ROS. In this study, we have shown for the first time that TCR cross-linking induced rapid (within 15 min) generation of both hydrogen peroxide and superoxide anion, as defined with oxidation-sensitive dyes, selective pharmacologic antioxidants, and overexpression of specific antioxidant enzymes. Furthermore, the data suggest the novel observation that superoxide anion and hydrogen peroxide are produced separately by distinct TCR-stimulated pathways. Unexpectedly, TCR-stimulated activation of the Fas ligand (FasL) promoter and subsequent cell death was dependent upon superoxide anion, but independent of hydrogen peroxide, while nuclear factor of activated T cells (NFAT) activation or interleukin 2 transcription was independent of all ROS. Anti-CD3 induced phosphorylation of extracellular signal–regulated kinase (ERK)1/2 required hydrogen peroxide generation but was unaffected by superoxide anion. Thus, antigen receptor signaling induces generation of discrete species of oxidants that selectively regulate two distinct redox sensitive pathways, a proapoptotic (FasL) and a proliferative pathway (ERK).

Fas-stimulated generation of reactive oxygen species or exogenous oxidative stress sensitize cells to Fas-mediated apoptosis.

Inhibition of Fas-mediated apoptosis in B cell lymphomas by thiol antioxidants (glutathione and N-acetylcysteine) supported previous studies, suggesting that Fas-stimulated ROS generation may play a role in Fas-mediated apoptosis. Thus, the goal of the current study was to determine if Fas stimulation could induce ROS generation and what role, if any, it played in apoptosis. Fas crosslinking induced rapid generation of ROS (within 15 min) well before the appearance of characteristic apoptotic changes. Overexpression of catalase or superoxide dismutase suggested that Fas induced production of both superoxide anion and hydrogen peroxide. ROS generation was only observed, however, in cells that were sensitive to apoptosis and not in B cells inherently resistant to anti-Fas or in those in which resistance was induced by B cell receptor crosslinking. The exogenous addition of 250 microM hydrogen peroxide could reverse the resistant phenotype and sensitize cells to Fas-induced apoptosis. In Fas-sensitive cells, depletion of endogenous antioxidant defenses with buthionine sulfoximine increased the sensitivity to Fas-induced apoptosis, while overexpression of antioxidant enzymes and antiapoptotic proteins suggested a role for Fas-induced production of hydrogen peroxide in apoptosis. Further analysis suggested a redox-sensitive step early in Fas signaling at the level of initiator caspase (caspase-8) activation. Thus, the data suggest that the level of oxidative stress, either from exogenous sources or generated endogenously upon receptor stimulation, regulates the sensitivity to Fas-mediated apoptosis.

T cell receptor-stimulated generation of hydrogen peroxide inhibits MEK-ERK activation and lck serine phosphorylation

Previous studies indicated that antigen receptor (TcR) stimulation of mature T cells induced rapid generation of reactive oxygen species (ROS). The goal of the current study was to examine the role(s) of ROS in TcR signal transduction, with a focus upon the redox-sensitive MAPK family. TcR cross-linking of primary human T blasts and Jurkat human T cells rapidly activated the ERK, JNK, p38 and Akt kinases within minutes, and was temporally associated with TcR-stimulated production of hydrogen peroxide (H(2)O(2)). TcR-induced activation of ERK was selectively augmented and sustained in the presence of pharmacologic antioxidants that can quench or inhibit H(2)O(2) production (NAC, MnTBAP and Ebselen, but not DPI), while activation of JNK and Akt were largely unaffected. This was paralleled by concurrent changes in MEK1/2 phosphorylation, suggesting that ROS acted upstream of MEK-ERK activation. Molecular targeting of H(2)O(2) by overexpression of peroxiredoxin II, a thioredoxin dependent peroxidase, also increased and sustained ERK and MEK activation upon TcR cross-linking. Enhancement of ERK phosphorylation by antioxidants correlated with increased and sustained serine phosphorylation of the src-family kinase lck, a known ERK substrate. Thus, the data suggest that TcR-stimulated production of hydrogen peroxide negatively feeds back to dampen antigen-stimulated ERK activation and this redox-dependent regulation may serve to modulate key steps in TcR signaling.

Abnormal Th1 Cell Differentiation and IFN-γ Production in T Lymphocytes from Motheaten Viable Mice Mutant for Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase-1

Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) plays an important role in T and B lymphocyte signaling; however, the function of SHP-1 in Th cell differentiation, in particular, the Th1 response, has not been defined. In this study, we provide evidence that SHP-1 phosphatase negatively regulates Th1 cell development and IFN-γ production. Compared with the wild-type control, anti-CD3-activated mouse T lymphocytes carrying the motheaten viable mutation in the SHP-1 gene produced a significantly increased amount of IFN-γ in the presence of IL-12. This increase was also seen at the basal level without IL-12 addition. Similarly, Th1 cell differentiation and proliferation of anti-CD3-activated SHP-1 mutant lymph node cells in the presence or absence of IL-12 were markedly enhanced, indicating a negative role for SHP-1 phosphatase in such lymphocyte activities. Interestingly, IL-12-induced activation of Jak2 and STAT4, critical components for IL-12-mediated cellular responses, was shortened or attenuated in mutant T cells. Together these results suggest that SHP-1 negatively regulates Th1 cell development and functions through a mechanism that is not directly related to IL-12 signaling.

B cell receptor signaling mediates immediate protection from Fas-induced apoptosis upstream of caspase activation through an atypical protein kinase C isozyme and de novo protein synthesis.

Signaling through the B cell receptor (BCR) of normal splenic B cells, as well as B cell lymphoma lines, can abrogate Fas‐mediated apoptosis. Using the B lymphoma line A20.2J, BCR signaling immediately inhibited Fas‐induced apoptosis upstream of caspase‐8 activation, as determined by Ile‐Glu‐Thr‐Asp‐(IETD)ase activity and cleavage of the caspase‐8 substrate Bid. Furthermore, following overexpression of a human Fas:FLICE construct, which directly induces caspase activation in a death‐inducing signaling complex‐independent manner, cells could not be protected through BCR stimulation.Co‐incubation with cycloheximide partially reversed protection from apoptosis and increased Fas‐stimulated initiator and effector caspase activation, suggesting new protein synthesis is necessary to induce protection upstream of caspase activation. Furthermore, co‐incubation with a broad‐spectrum protein kinase C (PKC) inhibitor, such as bisindolylmaleimide (Bis), also partially reversed protection from apoptosis, and examination of a panel of PKC inhibitors suggested a role for atypical isozymes in protection. Bis also acted to increase initiator and effector caspase activation upon anti‐IgG and anti‐Fas treatment. These data suggest that BCR‐induced protection is being mediated upstream of initiator caspase activation, and is partially dependent upon both PKC family members and new protein synthesis.

Overexpression of Insulin Receptor Substrate-1, But Not Insulin Receptor Substrate-2, Protects a T Cell Hybridoma from Activation-Induced Cell Death

The insulin receptor substrate (IRS) family of signaling molecules is expressed in lymphocytes, although their functions in these cells is largely unknown. To investigate the role of IRS in the protection of T cells from activation-induced cell death (AICD), we transfected the T cell hybridoma A1.1, which is IL-4 responsive but lacks expression of IRS family members with cDNA encoding IRS1 or IRS2. Stimulation of these clones with immobilized anti-CD3-induced expression of CD69 to the same level as the parental A1.1 cells. However, the A1.1 IRS1-expressing cells were markedly resistant to AICD, while the A1.1 IRS2-expressing cells were not. Inhibition of phosphatidylinositol 3′-kinase in the A1.1 IRS1-expressing cells did not abrogate their resistance to AICD. Fas mRNA was induced similarly by anti-CD3 in A1.1, A1.1 IRS1-expressing, and A1.1 IRS2-expressing cells. However, induction of Fas ligand (FasL) mRNA and functional FasL protein was delayed and decreased in IRS1-expressing cells, but not in IRS2-expressing cells. The induction of transcription from a 500-bp FasL promoter and a minimal 16-mer early growth response element linked to luciferase was also impaired in the IRS1-expressing cells. These results suggest that overexpression of IRS1, but not IRS2, protects A1.1 cells from AICD by diminishing FasL transcription through a pathway that is independent of the tyrosine phosphorylation of IRS1 and phosphatidylinositol 3′-kinase activity.