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1.
Cell Death Differ ; 21(6): 904-14, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24510126

ABSTRACT

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.


Subject(s)
Caspase 3/biosynthesis , Cell Survival/genetics , Interleukin-15/metabolism , T-Lymphocytes/metabolism , Animals , Apoptosis/genetics , Caspase 3/genetics , Glycolysis , Interleukin-15/genetics , Interleukin-2 , Lymphocyte Activation/genetics , Mice , Mitochondria/metabolism , Signal Transduction
2.
Cell Death Dis ; 4: e786, 2013 Sep 05.
Article in English | MEDLINE | ID: mdl-24008730

ABSTRACT

Mediators produced by the airway epithelium control the activation, recruitment, and survival of pulmonary dendritic cells (DC) that present antigen to CD4(+) T cells during the genesis and exacerbation of allergic asthma. The epithelial-derived acute phase protein, serum amyloid A (SAA), induces DC maturation and TH17 polarization. TH17 responses are associated with severe forms of allergic asthma that are poorly controlled by corticosteroids. We sought to determine whether SAA would enhance the survival of DC during serum starvation and could then contribute to the development of a glucocorticoid-resistant phenotype in CD4(+) T cells. Bone marrow-derived dendritic cells (BMDC) that were serum starved in the presence of SAA were protected from activation of caspase-3 and released less lactate dehydrogenase. In comparison with untreated serum-starved BMDC, treatment with SAA downregulated mRNA expression of the pro-apoptotic molecule Bim, increased production of the pro-survival heat shock protein 70 (HSP70), and induced secretion of pro-inflammatory cytokines. SAA-treated BMDC that were serum starved for 48 h remained capable of presenting antigen and induced OTII CD4(+) T cells to secrete IL-17A, IL-17F, IL-21, IL-22, and IFNγ in the presence of ovalbumin. IL-17A, IL-17F, IL-21, and IFNγ production occurred even when the CD4(+) T cells were treated with dexamethasone (Dex), whereas glucocorticoid treatment abolished cytokine secretion by T cells cocultured with untreated serum-starved BMDC. Measurement of Dex-responsive gene expression demonstrated CD4(+) T cells as the target of glucocorticoid hyperresponsiveness manifest as a consequence of BMDC stimulation by SAA. Finally, allergic airway disease induced by SAA and antigen inhalation was unresponsive to Dex treatment. Our results indicate that apo-SAA affects DC to both prolong their viability and increase their inflammatory potential under apoptosis-inducing conditions. These findings reveal mechanisms through which SAA enhances the CD4(+) T-cell-stimulating capacity of antigen-presenting cells that may actively participate in the pathogenicity of glucocorticoid-resistant lung disease.


Subject(s)
Apoptosis/drug effects , CD4-Positive T-Lymphocytes/cytology , Dendritic Cells/cytology , Glucocorticoids/pharmacology , Serum Amyloid A Protein/pharmacology , Animals , Apoptosis/immunology , Apoptosis Regulatory Proteins/metabolism , Bcl-2-Like Protein 11 , Bone Marrow Cells/cytology , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/immunology , Caspase 3/metabolism , Culture Media, Serum-Free , Cytokines/biosynthesis , Cytoprotection/drug effects , Dendritic Cells/drug effects , Dendritic Cells/enzymology , Dexamethasone/pharmacology , Down-Regulation/drug effects , Down-Regulation/genetics , Drug Resistance/drug effects , Enzyme Activation/drug effects , HSP70 Heat-Shock Proteins/metabolism , Hypersensitivity/immunology , Hypersensitivity/pathology , Immunization , Inflammation/pathology , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Proto-Oncogene Proteins/metabolism , Solubility
3.
Cell Death Differ ; 15(11): 1701-11, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18617900

ABSTRACT

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.


Subject(s)
Caspase 8/metabolism , Lymphocyte Activation/immunology , T-Lymphocytes/cytology , T-Lymphocytes/enzymology , CD3 Complex/metabolism , Cell Death , DNA Fragmentation , Enzyme Activation , Humans , Jurkat Cells , Kinetics , Membrane Microdomains/enzymology , Protein Transport , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/immunology , fas Receptor/metabolism
4.
J Immunol ; 167(11): 6654-62, 2001 Dec 01.
Article in English | MEDLINE | ID: mdl-11714837

ABSTRACT

Ag stimulation of CD8+ lymphocytes in vivo results in their migration to various tissues as well as the activation of a cytolytic program involving perforin, TNF-alpha, and Fas ligand. The liver is one of the main sites for infiltration by activated CD8+ T cells, and this is followed by the death of hepatocytes. The contribution of the various cytolytic components to this process is unclear. Hepatocyte damage by CD8+ T cells was studied using the MHC class I-restricted OVA-specific TCR transgenic mouse (OT-1) to examine the contribution of Fas to hepatocyte death. Activated CD8+ T cells from both OT-1 and Fas-deficient OT-1lpr mice migrated to the liver in similar numbers after OVA administration, but only in OT-1 mice was there evidence of significant hepatocyte damage histologically and by elevation of serum aspartate transaminase. These differences were not the result of inefficient induction of cytolytic activity in OT-1lpr liver T cells, since they were as cytolytic in vitro as OT-1 liver T cells. This was supported by findings of similar high levels of message for perforin, TNF-alpha, and Fas ligand in liver lymphocytes from both mice. These findings demonstrate that following Ag activation, infiltrating liver CD8+ T lymphocytes induce hepatocyte damage in a Fas-dependent manner.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Cell Movement/immunology , Cytotoxicity, Immunologic/immunology , Liver/immunology , Liver/pathology , fas Receptor/physiology , Animals , Cell Death/immunology , Egg Proteins/administration & dosage , Egg Proteins/immunology , Female , Hepatocytes/immunology , Hepatocytes/pathology , Immunophenotyping , Injections, Intraperitoneal , Male , Mice , Mice, Inbred C57BL , Mice, Inbred MRL lpr , Mice, Transgenic , Ovalbumin/administration & dosage , Ovalbumin/immunology , Peptide Fragments/administration & dosage , Peptide Fragments/immunology , Species Specificity , Tumor Cells, Cultured , fas Receptor/toxicity
5.
Curr Opin Immunol ; 13(3): 356-62, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11406369

ABSTRACT

The death of T lymphocytes following their activation involves several signal pathways that converge on a series of proteases, known as caspases, that degrade cellular proteins and activate a DNAse. Caspases are activated through ligation of cell surface death receptors as well as via direct activation of downstream caspases, often through metabolic stress such as cytokine withdrawal or generation of oxygen radicals, that culminates in mitochondrial dysfunction and release of the pro-apoptotic molecules, cytochrome c and Smac/DIABLO. The Bcl-2 family members serve to regulate the mitochondrial membrane integrity. Recent studies are now revealing the significant contribution to the activation-induced cell death of T cells by downstream caspases such as caspase-3 and Bcl-2-homology domain 3 (BH3)-only members of the Bcl-2 family.


Subject(s)
Apoptosis/immunology , Lymphocyte Activation/physiology , Animals , Humans , Receptors, Tumor Necrosis Factor/physiology , Signal Transduction/immunology , T-Lymphocytes/cytology , T-Lymphocytes/enzymology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , fas Receptor/physiology
6.
Curr Biol ; 10(11): 640-8, 2000 Jun 01.
Article in English | MEDLINE | ID: mdl-10837247

ABSTRACT

BACKGROUND: Activation of Fas (CD95) by its ligand (FasL) rapidly induces cell death through recruitment and activation of caspase-8 via the adaptor protein Fas-associated death domain protein (FADD). However, Fas signals do not always result in apoptosis but can also trigger a pathway that leads to proliferation. We investigated the level at which the two conflicting Fas signals diverge and the protein(s) that are implicated in switching the response. RESULTS: Under conditions in which proliferation of CD3-activated human T lymphocytes is increased by recombinant FasL, there was activation of the transcription factors NF-kappaB and AP-1 and recruitment of the caspase-8 inhibitor and FADD-interacting protein FLIP (FLICE-like inhibitory protein). Fas-recruited FLIP interacts with TNF-receptor associated factors 1 and 2, as well as with the kinases RIP and Raf-1, resulting in the activation of the NF-kappaB and extracellular signal regulated kinase (Erk) signaling pathways. In T cells these two signal pathways are critical for interleukin-2 production. Increased expression of FLIP in T cells resulted in increased production of interleukin-2. CONCLUSIONS: We provide evidence that FLIP is not simply an inhibitor of death-receptor-induced apoptosis but that it also mediates the activation of NF-kappaB and Erk by virtue of its capacity to recruit adaptor proteins involved in these signaling pathways.


Subject(s)
Carrier Proteins/metabolism , Caspase Inhibitors , Intracellular Signaling Peptides and Proteins , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Signal Transduction/physiology , T-Lymphocytes/physiology , Apoptosis , CASP8 and FADD-Like Apoptosis Regulating Protein , CD3 Complex/physiology , Caspase 8 , Caspase 9 , Cells, Cultured , Fas Ligand Protein , Humans , Interleukin-2/biosynthesis , Membrane Glycoproteins/pharmacology , Proteins/metabolism , Proto-Oncogene Proteins c-raf , Receptor-Interacting Protein Serine-Threonine Kinases , Receptors, Tumor Necrosis Factor/physiology , Signal Transduction/drug effects , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , TNF Receptor-Associated Factor 1 , TNF Receptor-Associated Factor 2 , Transcription Factor AP-1/metabolism , fas Receptor/physiology
7.
J Exp Med ; 190(12): 1891-6, 1999 Dec 20.
Article in English | MEDLINE | ID: mdl-10601363

ABSTRACT

Triggering of Fas (CD95) by its ligand (FasL) rapidly induces cell death via recruitment of the adaptor protein Fas-associated death domain (FADD), resulting in activation of a caspase cascade. It was thus surprising that T lymphocytes deficient in FADD were reported recently to be not only resistant to FasL-mediated apoptosis, but also defective in their proliferative capacity. This finding suggested potentially dual roles of cell growth and death for Fas and possibly other death receptors. We report here that CD3-induced proliferation and interleukin 2 production by human T cells are blocked by inhibitors of caspase activity. This is paralleled by rapid cleavage of caspase-8 after CD3 stimulation, but no detectable processing of caspase-3 during the same interval. The caspase contribution to T cell activation may occur via TCR-mediated upregulation of FasL, as Fas-Fc blocked T cell proliferation, whereas soluble FasL augmented CD3-induced proliferation. These findings extend the role of death receptors to the promotion of T cell growth in a caspase-dependent manner.


Subject(s)
Caspases/immunology , Signal Transduction/immunology , T-Lymphocytes/cytology , T-Lymphocytes/immunology , CD3 Complex/immunology , Cell Division/immunology , Enzyme Activation/immunology , Fas Ligand Protein , Humans , Lymphocyte Activation , Membrane Glycoproteins/immunology , fas Receptor/immunology
8.
Cell Immunol ; 194(1): 67-77, 1999 May 25.
Article in English | MEDLINE | ID: mdl-10357882

ABSTRACT

Although the protease cascade initiated by Fas (CD95, Apo-1) is well characterized, there remains little known about how kinase pathways may impact on Fas-mediated apoptosis. We recently observed that in T lymphocytes Fas strongly induced activation of JNK (c-Jun N-terminal kinase) but not of second messengers leading to activation of ERK (extracellular regulated kinase). Additionally, Fas-mediated apoptosis was significantly inhibited with PMA, a potent activator of the ERK signaling pathway. This suggested a model whereby activation of the ERK pathway might attenuate Fas-mediated apoptosis. This was confirmed in the current study by showing that activation of MEK1, the upstream regulator of ERK, reduces Fas-mediated apoptosis, whereas inhibition of MEK1 augments apoptosis by Fas. Furthermore, Fas-mediated apoptosis of Jurkat T cells is not affected by constitutively active or dominant negative variants that modulate the JNK pathway. These results demonstrate that Fas-induced JNK activation is not required for apoptosis by Jurkat T cells, but rather is more likely secondary to cell stress during the early phases of apoptosis. This is supported by the ability of the caspase blocker zVAD to inhibit both apoptosis and JNK activation by Fas.


Subject(s)
Apoptosis , Mitogen-Activated Protein Kinase Kinases , Mitogen-Activated Protein Kinases , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , T-Lymphocytes/enzymology , fas Receptor/metabolism , Annexin A5/metabolism , Biomarkers , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Enzyme Activation , Humans , JNK Mitogen-Activated Protein Kinases , Jurkat Cells , Kinetics , MAP Kinase Kinase 1 , Mitogen-Activated Protein Kinase 9 , Protein Kinases/metabolism , Staining and Labeling , T-Lymphocytes/pathology
9.
J Immunol ; 162(10): 5747-56, 1999 May 15.
Article in English | MEDLINE | ID: mdl-10229807

ABSTRACT

The origin of TCR-alphabeta+ CD4-CD8- cells is unclear, yet accumulating evidence suggests that they do not represent merely a default pathway of unselected thymocytes. Rather, they arise by active selection as evidenced by their absence in mice lacking expression of class I MHC. TCR-alphabeta+ CD4-CD8- cells also preferentially accumulate in mice lacking expression of Fas/APO-1/CD95 (lpr) or Fas-ligand (gld), suggesting that this subset might represent a subpopulation destined for apoptosis in normal mice. Findings from mice bearing a self-reactive TCR transgene support this view. In the current study we observe that in normal mice, TCR-alphabeta+ CD4-CD8- thymocytes contain a high proportion of cells undergoing apoptosis. The apoptotic subpopulation is further identified by its expression of B220 and IL2Rbeta and the absence of surface CD2. The CD4-CD8- B220+ phenotype is also enriched in T cells that recognize endogenous retroviral superantigens, and can be induced in TCR transgenic mice using peptide/MHC complexes that bear high affinity, but not low affinity, for TCR. A model is presented whereby the TCR-alphabeta+ CD2- CD4-CD8- B220+ phenotype arises from high intensity TCR signals. This model is broadly applicable to developing thymocytes as well as mature peripheral T cells and may represent the phenotype of self-reactive T cells that are increased in certain autoimmune conditions.


Subject(s)
CD4 Antigens/isolation & purification , CD8 Antigens/isolation & purification , Leukocyte Common Antigens/isolation & purification , Receptors, Antigen, T-Cell, alpha-beta/isolation & purification , T-Lymphocyte Subsets/immunology , Amino Acid Sequence , Animals , Apoptosis , CD2 Antigens/isolation & purification , Major Histocompatibility Complex/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred MRL lpr , Mice, Transgenic , Models, Immunological , Molecular Sequence Data , Ovalbumin/immunology , Peptide Fragments/immunology , Phenotype , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Interleukin-2/isolation & purification , Signal Transduction , Thymus Gland/cytology , Thymus Gland/immunology
10.
Ann N Y Acad Sci ; 887: 181-90, 1999.
Article in English | MEDLINE | ID: mdl-10668474

ABSTRACT

Group B coxsackieviruses (CVB), which infect the myocardium, cause myocarditis and dilated cardiomyopathy. However, not all infections of the myocardium result in disease. In the mouse model, CVB infection stimulates autoimmune T cell response to cardiac antigens, and these autoimmune effectors cause myocyte necrosis and cardiomyopathy. Induction of pathogenic autoimmunity depends upon CD4+ Th1 (interferon-gamma positive) cells while Th2 (IL-4 positive) cell responses promote disease resistance. T lymphocytes expressing the gamma-delta T cell receptor (gamma delta +) constitute up to 12% of the inflammatory cells in the heart and are crucial to maintaining a dominant Th1 response phenotype. gamma delta + lymphocytes modulate T cell responses by selectively lysing CD4+ Th2 cells. Th1 cells are not killed by gamma delta + cells. Lysis requires direct cell:cell interaction between the gamma delta + cell and CD4+ Th2 target and is most likely mediated through Fas:FasL interaction. These studies demonstrate a novel mechanism for immune modulation of cytokine responses in vivo.


Subject(s)
Apoptosis , Cardiomyopathy, Dilated/immunology , Coxsackievirus Infections/immunology , Coxsackievirus Infections/pathology , Enterovirus B, Human , Myocarditis/immunology , T-Lymphocytes/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , Cardiomyopathy, Dilated/pathology , Cardiomyopathy, Dilated/virology , Humans , Inflammation , Mice , Mice, Inbred BALB C , Myocarditis/pathology , Myocarditis/virology , Receptors, Antigen, T-Cell, gamma-delta/immunology , Th1 Cells/immunology , Th2 Cells/immunology
11.
J Immunol ; 161(10): 5762-71, 1998 Nov 15.
Article in English | MEDLINE | ID: mdl-9820558

ABSTRACT

Lyme arthritis synovial fluid contains a large proportion of gamma delta T cells that proliferates upon stimulation with the causative spirochete, Borrelia burgdorferi. A panel of Borrelia-reactive gamma delta T cell clones was derived from synovial fluid of two patients with Lyme arthritis. Each of six gamma delta clones from one patient used the V delta 1 TCR segment but had otherwise unique CDR3 sequences and diverse V gamma segment usage. Stimulation of the V delta 1 clones was optimal in the presence of Borrelia, dendritic cells, and exogenous IL-2, which was reflected by proliferation, TCR down-modulation, as well as induction of CD25 and Fas ligand expression. Stimulation by B. burgdorferi-pulsed dendritic cells withstood chemical fixation and was not restricted to class I or class II MHC, CD1a, CD1b, or CD1c. In contrast, anti-gamma delta antibody potently inhibited proliferation. Extraction of B. burgdorferi lipoproteins with Triton X-114 enriched for the stimulatory component. This was confirmed using lipidated vs nonlipidated hexapeptides of Borrelia outer surface proteins. These observations suggest that synovial V delta 1 T cells may mediate an innate immune response to common lipoprotein products of spirochetes.


Subject(s)
Borrelia burgdorferi Group/immunology , Lipoproteins/immunology , Lyme Disease/immunology , Oligopeptides/immunology , Receptors, Antigen, T-Cell, gamma-delta/immunology , Synovial Fluid/immunology , T-Lymphocyte Subsets/immunology , Adolescent , Amino Acid Sequence , Antibodies, Monoclonal/pharmacology , Bacterial Proteins/immunology , Base Sequence , Child , Clone Cells/immunology , Clone Cells/microbiology , Dendritic Cells/immunology , Dendritic Cells/microbiology , Female , Fixatives , Humans , Immunosuppressive Agents/pharmacology , Lyme Disease/microbiology , Lymphocyte Activation/genetics , Major Histocompatibility Complex/genetics , Molecular Sequence Data , Mycobacterium/immunology , Oligopeptides/metabolism , Spirochaetales/immunology , Synovial Fluid/cytology , Synovial Fluid/microbiology , T-Lymphocyte Subsets/microbiology
12.
J Exp Med ; 188(6): 1147-57, 1998 Sep 21.
Article in English | MEDLINE | ID: mdl-9743533

ABSTRACT

Little is understood of the anatomical fate of activated T lymphocytes and the consequences they have on the tissues into which they migrate. Previous work has suggested that damaged lymphocytes migrate to the liver. This study compares class I versus class II major histocompatibility complex (MHC)-restricted ovalbumin-specific T cell antigen receptor (TCR) transgenic mice to demonstrate that after in vivo activation with antigen the emergence of CD4(-)CD8(-)B220(+) T cells occurs more frequently from a CD8(+) precursor than from CD4(+) T cells. Furthermore, this change in phenotype is conferred only by the high affinity native peptide antigen and not by lower affinity peptide variants. After activation of CD8(+) cells with only the high affinity peptide, there is also a dramatically increased number of liver lymphocytes with accompanying extensive hepatocyte damage and elevation of serum aspartate transaminase. This was not observed in mice bearing a class II MHC-restricted TCR. The findings show that CD4(-)CD8(-)B220(+) T cells preferentially derive from a CD8(+) precursor after a high intensity TCR signal. After activation, T cells can migrate to the liver and induce hepatocyte damage, and thereby serve as a model of autoimmune hepatitis.


Subject(s)
Antigens/pharmacology , CD8-Positive T-Lymphocytes/immunology , Liver/pathology , Ovalbumin/immunology , Peptides/immunology , Amino Acid Sequence , Animals , Antigens/administration & dosage , CD4 Antigens/biosynthesis , CD8 Antigens/biosynthesis , CD8-Positive T-Lymphocytes/metabolism , Cell Death/immunology , Cell Movement/immunology , Cell Size/immunology , Female , Humans , Immunophenotyping , Injections, Intraperitoneal , Leukocyte Common Antigens/biosynthesis , Liver/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Molecular Sequence Data , Ovalbumin/administration & dosage , Peptides/administration & dosage
13.
J Immunol ; 161(2): 729-39, 1998 Jul 15.
Article in English | MEDLINE | ID: mdl-9670949

ABSTRACT

The CD40/CD40L (CD40 ligand) axis regulates several interactions between T cells and B cells. Blocking of CD40 engagement by CD40L inhibits Ig class switch by B cells as well as diminishes T cell response to an immunizing Ag. For these reasons, disruption of CD40/CD40L interactions by anti-CD40L administration or by genetic disruption of CD40L has ameliorated a variety of autoimmune conditions. More recent findings suggest that a direct signal can be transmitted to T cells via their expressed CD40L, which can costimulate proliferation with CD3 or promote germinal center formation. It is therefore possible that treatment with anti-CD40L Ab might produce a different outcome than observed in genetically CD40L-deficient mice. In this regard, we observe that in contrast to the genetic deletion of CD40L in MRL-lpr mice, which diminishes autoimmune disease but has little effect on adenopathy, administration of anti-CD40L to MRL-lpr mice accelerates both of these parameters. This difference appears to result from anti-CD40L actively delivering a signal that inhibits T cell apoptosis in lpr mice. This was confirmed by in vitro studies demonstrating that CD40L cross-linking on lpr thymocytes inhibited apoptosis and surface TCR down-modulation induced by CD3 ligation.


Subject(s)
Antibodies, Monoclonal/administration & dosage , Apoptosis/immunology , CD40 Antigens/immunology , Glomerulonephritis/immunology , Lymphatic Diseases/immunology , Membrane Glycoproteins/immunology , T-Lymphocyte Subsets/immunology , Animals , Autoantibodies/biosynthesis , CD40 Antigens/biosynthesis , CD40 Ligand , Female , Glomerulonephritis/etiology , Glomerulonephritis/mortality , Injections, Intraperitoneal , Ligands , Lymph Nodes/cytology , Lymph Nodes/metabolism , Lymphatic Diseases/etiology , Mice , Mice, Inbred MRL lpr , Receptors, Antigen, T-Cell, alpha-beta , Survival Analysis , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/metabolism , Thymus Gland/cytology , Thymus Gland/metabolism
14.
J Immunol ; 160(10): 4881-8, 1998 May 15.
Article in English | MEDLINE | ID: mdl-9590235

ABSTRACT

Fas (Apo-1, CD95), a member of the TNFR family, is expressed on a variety of cell types and transduces an apoptotic signal. Since Fas does not possess known enzymatic activities, proteins that interact with the cytoplasmic domain of Fas regulate the death signal. Several proteins have been identified, primarily using the yeast two-hybrid system, that associate with the death domain of Fas. One of these proteins, FADD/MORT1, can be phosphorylated, although the kinase that is responsible has not been identified. Furthermore, direct signaling connections between Fas and its known activation of sphingomyelinase or NF-kappaB have not been made, suggesting that other proteins may associate with Fas. In this study, a series of glutathione S-transferase fusion proteins was constructed that contained the cytoplasmic domain of murine Fas. These proteins were used to search for additional proteins that associate with Fas. Novel proteins, including kinases, were identified that associated specifically with the membrane-proximal, cytoplasmic tail of Fas but not with the death domain. One of these kinases phosphorylates FADD/MORT1. Moreover, the membrane-proximal region of Fas itself was phosphorylated by one of the associating kinases. These findings suggest that, similar to the Fas-related p55 TNFR, the membrane-proximal region of Fas likely participates in signaling.


Subject(s)
Adaptor Proteins, Signal Transducing , Carrier Proteins/metabolism , Protein Kinases/metabolism , fas Receptor/metabolism , Animals , Cytoplasm/metabolism , Fas-Associated Death Domain Protein , Mice , Molecular Weight , Myelin Basic Protein/metabolism , Phosphorylation , Tumor Cells, Cultured
15.
Eur J Immunol ; 28(1): 70-9, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9485187

ABSTRACT

CD2 is a cell surface glycoprotein expressed on most T lymphocytes that is generally viewed as a cell adhesion molecule and, in this capacity, contributes to T cell receptor (TCR) signaling. CD2 has a relatively long cytoplasmic tail which associates with the src family tyrosine kinases, p56(lck) and p59(fyn), and could potentially signal directly. Down-modulation of CD2 on T cells has been shown to result in diminished proliferative capacity and interleukin (IL)-2 production. Furthermore, re-expression of CD2 can result in the restoration of these functions. This suggests that CD2 can influence the intensity of TCR signaling. As TCR signal intensity is pivotal to the induction of T cell apoptosis, we considered the hypothesis that the level of CD2 on the T cell surface may influence its propensity toward apoptosis. Using an anti-CD2 antibody, CD2 was down-modulated in vivo on mouse T lymphocytes without affecting the levels of surface CD3, TCR alphabeta, CD4 or CD8. Deletion of superantigen-responsive T cells was delayed in mice with down-modulated CD2 following the administration of staphylococcal enterotoxin B (SEB). This was paralleled by diminished apoptosis of SEB-responsive cells. The findings suggest a model whereby the level of CD2 expression influences the intensity of TCR signaling and the ability to undergo apoptosis.


Subject(s)
Antigens, Bacterial/immunology , CD2 Antigens/physiology , CD4-Positive T-Lymphocytes/immunology , Clonal Deletion , Down-Regulation/drug effects , Enterotoxins/immunology , Lymphocyte Activation/drug effects , Superantigens/immunology , Animals , Antigens, Surface/analysis , CD2 Antigens/biosynthesis , CD4-Positive T-Lymphocytes/drug effects , Gene Rearrangement, beta-Chain T-Cell Antigen Receptor , Immune Tolerance , Interleukin-2/biosynthesis , Mice , Mice, Inbred BALB C , Signal Transduction/drug effects
16.
Infect Immun ; 66(3): 1092-9, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9488400

ABSTRACT

A common concern with many autoimmune diseases of unknown etiology is the extent to which tissue T-lymphocyte infiltrates, versus a nonspecific infiltrate, reflect a response to the causative agent. Lyme arthritis can histologically resemble rheumatoid synovitis, particularly the prominent infiltration by T lymphocytes. This has raised speculation about whether Lyme synovitis represents an ongoing response to the causative spirochete, Borrelia burgdorferi, or rather a self-perpetuating autoimmune reaction. In an effort to answer this question, the present study examined the repertoire of infiltrating T cells in synovial fluid from nine Lyme arthritis patients, before and after stimulation with B. burgdorferi. Using a highly sensitive and consistent quantitative PCR technique, a comparison of the T-cell antigen receptor (TCR) beta-chain variable (Vbeta) repertoires of the peripheral blood and synovial fluid showed a statistically significant increase in expression of Vbeta2 and Vbeta6 in the latter. This is remarkably similar to our previous findings in studies of rheumatoid arthritis and to other reports on psoriatic skin lesions. However, stimulation of synovial fluid T cells with B. burgdorferi provoked active proliferation but not a statistically significant increase in expression of any TCR Vbeta, including Vbeta2 and Vbeta6. Collectively, the findings suggest that the skewing of the TCR repertoire of fresh synovial fluid in Lyme arthritis may represent more a synovium-tropic or nonspecific inflammatory response, similar to that occurring in rheumatoid arthritis or psoriasis, rather than a specific Borrelia reaction.


Subject(s)
Lyme Disease/immunology , Receptors, Antigen, T-Cell, alpha-beta/analysis , Adolescent , Aged , Child , Female , HLA-DR Antigens/analysis , HLA-DR Antigens/genetics , Humans , Lymphocyte Activation , Male , Middle Aged , Polymerase Chain Reaction , Synovial Fluid/immunology
17.
Coron Artery Dis ; 8(10): 593-7, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9457441

ABSTRACT

The concept that cells might possess the capacity to self-destruct by a process of programmed cell death, or apoptosis, is rather recent. Interest in this possibility suddenly galvanized when the first death genes were described in round worms 10 years ago. This led to novel rethinking of many disease processes, such as cancer, which might not purely be due to uncontrolled proliferation, but also to the lack of necessary death of lymphocytes or in autoimmune diseases with the lack of removal of self-reactive lymphocytes. This overview chronicles the events leading up to the paradigm of cell death as an active process. The commonly-used assays for measuring apoptosis are described and compared. Examples of the need for apoptosis are highlighted. This is followed by a discussion of how apoptosis is initiated in a variety of systems and what signal pathways are used, providing suggestions as to how the process of apoptosis might be therapeutically manipulated.


Subject(s)
Apoptosis/physiology , Apoptosis/genetics , Cardiovascular Diseases/etiology , Cardiovascular Diseases/physiopathology , Humans
18.
J Exp Med ; 184(6): 2109-17, 1996 Dec 01.
Article in English | MEDLINE | ID: mdl-8976167

ABSTRACT

The function of the minor subset of T lymphocytes bearing the gamma delta T cell antigen receptor is uncertain. Although some gamma delta T cells react to microbial products, responsiveness has only rarely been demonstrated toward a bacterial antigen from a naturally occurring human infection. Synovial fluid lymphocytes from patients with Lyme arthritis contain a large proportion of gamma delta cells that proliferate in response to the causative spirochete, Borrelia burgdorferi. Furthermore, synovial gamma delta T cell clones express elevated and sustained levels of the ligand for Fas (APO-1, CD95) compared to alpha beta T cells, and induce apoptosis of Fashigh CD4+ synovial lymphocytes. The findings suggest that gamma delta T cells contribute to defense in human infections, as well as manifest an immunoregulatory function at inflammatory sites by a Fas-dependent process.


Subject(s)
Apoptosis , Arthritis, Infectious/immunology , Borrelia burgdorferi Group/immunology , CD4-Positive T-Lymphocytes/immunology , Lyme Disease/immunology , Receptors, Antigen, T-Cell, gamma-delta/immunology , Synovial Fluid/immunology , T-Lymphocyte Subsets/immunology , fas Receptor , CD4-Positive T-Lymphocytes/microbiology , CD8-Positive T-Lymphocytes/immunology , Clone Cells , DNA Primers , Flow Cytometry , Humans , Immunophenotyping , Lymphocyte Activation , Polymerase Chain Reaction , Receptors, Antigen, T-Cell, gamma-delta/analysis , Receptors, Antigen, T-Cell, gamma-delta/biosynthesis
19.
Eur J Immunol ; 26(12): 2952-63, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8977291

ABSTRACT

CD2 is a cell surface glycoprotein present on all T cells which has been shown to function as an adhesion and signaling molecule. Expressed early in T cell development, human CD2 (HCD2) has been suggested to play a role during thymopoiesis. However, the relevance of CD2 in T cell development has been called into question recently, as neither disruption of the CD2 gene nor anti-CD2 antibody treatment of fetal thymic organ cultures in mouse were shown to have any discernible consequences. We have expressed HCD2 at high levels in transgenic mice and found a profound effect of the transgene on thymocyte differentiation. Transgenic thymuses are considerably reduced in cell number as a consequence of increased apoptosis of double-positive (DP) thymocytes in the cortex. The remaining DP cells have up-regulated levels of T cell receptor (TCR) and are resistant to apoptosis mediated by administration of antigen. These effects are dependent on the cytoplasmic domain of HCD2, as mice expressing comparable levels of a tailless HCD2 transgene have a normal phenotype. The HCD2 cytoplasmic domain contains several regions of identity with mouse CD2 and can interact effciently with mouse intracellular signaling machinery. These results suggest there is considerable cross-talk between CD2 and TCR on developing thymocytes with consequences for the stimulation threshold of mature T cells.


Subject(s)
CD2 Antigens/genetics , CD2 Antigens/physiology , Down-Regulation/drug effects , Signal Transduction/genetics , Signal Transduction/immunology , Thymus Gland/metabolism , Transgenes/genetics , Animals , Antigens, CD/drug effects , Apoptosis/drug effects , CD48 Antigen , Clonal Deletion/drug effects , Humans , Lymphocyte Specific Protein Tyrosine Kinase p56(lck) , Mice , Mice, Transgenic , Receptor-CD3 Complex, Antigen, T-Cell/biosynthesis , Thymus Gland/cytology , src-Family Kinases/drug effects
20.
Eur J Immunol ; 26(5): 989-94, 1996 May.
Article in English | MEDLINE | ID: mdl-8647190

ABSTRACT

Fas is a cell surface molecule that is expressed on a wide array of cell types and triggers apoptosis. While in most situations Fas ligation activates programmed cell death, on resting T lymphocytes it can co-stimulate proliferation with the T cell receptor (TCR)/CD3 complex. This incongruity suggests that Fas may elicit signaling events that overlap with those used by proliferation cues. We observe that in the human T cell line Jurkat and in human peripheral blood lymphocytes, Fas stimulation does not signal by the Ras/Raf-1/mitogen-activated protein kinase (MAPK) pathway or by increased intracellular calcium. Rather, Fas ligation strongly activates Jun kinase (JNK). This activity, as well as Fas-induced apoptosis, is blocked by increased levels of cAMP. The balance between proliferation and apoptosis by Fas triggering of T lymphocytes may therefore reflect a signaling ratio between TCR activation of the Ras/Raf-1/MAPK pathway versus JNK activation by Fas.


Subject(s)
Apoptosis/immunology , Calcium-Calmodulin-Dependent Protein Kinases/biosynthesis , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinase Kinases , Protein Kinases/biosynthesis , T-Lymphocytes/enzymology , T-Lymphocytes/immunology , fas Receptor/physiology , Cell Line , Enzyme Activation/immunology , Humans , Interleukin-2/biosynthesis , Lymphocyte Activation , Lymphoma , MAP Kinase Kinase 4 , Signal Transduction/immunology , T-Lymphocytes/metabolism , Tumor Cells, Cultured
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