Mitochondrial control of calcium-channel gating: A mechanism for sustained signaling and transcriptional activation in T lymphocytes

In addition to their well-known functions in cellular energy transduction, mitochondria play an important role in modulating the amplitude and time course of intracellular Ca2+ signals. In many cells, mitochondria act as Ca2+ buffers by taking up and releasing Ca2+, but this simple buffering action by itself often cannot explain the organelle's effects on Ca2+ signaling dynamics. Here we describe the functional interaction of mitochondria with store-operated Ca2+ channels in T lymphocytes as a mechanism of mitochondrial Ca2+ signaling. In Jurkat T cells with functional mitochondria, prolonged depletion of Ca2+ stores causes sustained activation of the store-operated Ca2+ current, ICRAC (CRAC, Ca2+ release-activated Ca2+). Inhibition of mitochondrial Ca2+ uptake by compounds that dissipate the intramitochondrial potential unmasks Ca2+-dependent inactivation of ICRAC. Thus, functional mitochondria are required to maintain CRAC-channel activity, most likely by preventing local Ca2+ accumulation near sites that govern channel inactivation. In cells stimulated through the T-cell antigen receptor, acute blockade of mitochondrial Ca2+ uptake inhibits the nuclear translocation of the transcription factor NFAT in parallel with CRAC channel activity and [Ca2+]i elevation, indicating a functional link between mitochondrial regulation of ICRAC and T-cell activation. These results demonstrate a role for mitochondria in controlling Ca2+ channel activity and signal transmission from the plasma membrane to the nucleus.

PU.1 as an essential activator for the expression of gp91phox gene in human peripheral neutrophils, monocytes, and B lymphocytes

We have reported a deficiency of a 91-kDa glycoprotein component of the phagocyte NADPH oxidase (gp91phox) in neutrophils, monocytes, and B lymphocytes of a patient with X chromosome-linked chronic granulomatous disease. Sequence analysis of his gp91phox gene revealed a single-base mutation (C → T) at position −53. Electrophoresis mobility-shift assays showed that both PU.1 and hematopoietic-associated factor 1 (HAF-1) bound to the inverted PU.1 consensus sequence centered at position −53 of the gp91phox promoter, and the mutation at position −53 strongly inhibited the binding of both factors. It was also indicated that a mutation at position −50 strongly inhibited PU.1 binding but hardly inhibited HAF-1 binding, and a mutation at position −56 had an opposite binding specificity for these factors. In transient expression assay using HEL cells, which express PU.1 and HAF-1, the mutations at positions −53 and −50 significantly reduced the gp91phox promoter activity; however, the mutation at position −56 did not affect the promoter activity. In transient cotransfection study, PU.1 dramatically activated the gp91phox promoter in Jurkat T cells, which originally contained HAF-1 but not PU.1. In addition, the single-base mutation (C → T) at position −52 that was identified in a patient with chronic granulomatous disease inhibited the binding of PU.1 to the promoter. We therefore conclude that PU.1 is an essential activator for the expression of gp91phox gene in human neutrophils, monocytes, and B lymphocytes.

Tyrosine kinase-dependent activation of a chloride channel in CD95-induced apoptosis in T lymphocytes

CD95/Fas/APO-1 mediated apoptosis is an important mechanism in the regulation of the immune response. Here, we show that CD95 receptor triggering activates an outwardly rectifying chloride channel (ORCC) in Jurkat T lymphocytes. Ceramide, a lipid metabolite synthesized upon CD95 receptor triggering, also induces activation of ORCC in cell-attached patch clamp experiments. Activation is mediated by Src-like tyrosine kinases, because it is abolished by the tyrosine kinase inhibitor herbimycin A or by genetic deficiency of p56lck. In vitro incubation of excised patches with purified p56lck results in activation of ORCC, which is partially reversed upon addition of anti-phosphotyrosine antibody. Inhibition of ORCC by four different drugs correlates with a 30–65% inhibition of apoptosis. Intracellular acidification observed upon CD95 triggering is abolished by inhibition of either ORCC or p56lck. The results suggest that tyrosine kinase-mediated activation of ORCC may play a role in CD95-induced cell death in T lymphocytes.

B lymphocyte involvement in ankylosing spondylitis: the heavy chain variable segment gene repertoire of B lymphocytes from germinal center-like foci in the synovial membrane indicates antigen selection

The synovial membrane (SM) of affected joints in ankylosing spondylitis (AS) is infiltrated by germinal center-like aggregates (foci) of lymphocytes similar to rheumatoid arthritis (RA). We characterized the rearranged heavy chain variable segment (VH) genes in the SM for gene usage and the mutational pattern to elucidate the B lymphocyte involvement in AS.

Induction of "tissue" transglutaminase in HIV pathogenesis: evidence for high rate of apoptosis of CD4+ T lymphocytes and accessory cells in lymphoid tissues.

Peripheral blood mononuclear cells and lymphoid tissues from HIV-infected individuals display high levels of "tissue" transglutaminase (tTG) with respect to seronegative persons. In asymptomatic individuals, > 80% of the circulating CD4+ T cells synthesize tTG protein and the number of these cells matches the level of apoptosis detected in the peripheral blood mononuclear cells from the same patients. In HIV-infected lymph nodes tTG protein is localized in large number of cells (macrophages, follicular dendritic cells, and endothelial cells), showing distinctive morphological and biochemical features of apoptosis as well as in lymphocytes and syncytia. These findings demonstrate that during the course of HIV infection, high levels of apoptosis also occur in the accessory cells of lymphoid organs. The increased concentration of epsilon(gamma-glutamyl)lysine isodipeptide, the degradation product of tTG cross-linked proteins, observed in the blood of HIV-infected individuals demonstrates that the enzyme accumulated in the dying cells actively cross-links intracellular proteins. The enhanced levels of epsilon(gamma-glutamyl)lysine in the blood parallels the progression of HIV disease, suggesting that the isodipeptide determination might be a useful method to monitor the in vivo rate of apoptosis.

Activation of Jak/STAT proteins involved in signal transduction pathway mediated by receptor for interleukin 2 in malignant T lymphocytes derived from cutaneous anaplastic large T-cell lymphoma and Sezary syndrome.

Signaling through the interleukin 2 receptor (IL-2R) involves phosphorylation of several proteins including Jak3, STAT5, and, in preactivated cells, STAT3. In the present study, we examined the functional status of the IL-2R-associated Jak/STAT pathway in malignant T lymphocytes from advanced skin-based lymphomas: anaplastic large T-cell lymphoma (ALCL) and Sezary syndrome (SzS). Proliferation of three ALCL cell lines (PB-1, 2A, and 2B) was partially inhibited by rapamycin, a blocker of some of the signals mediated by IL-2R, but not by cyclosporin A, FK-506, and prednisone, which suppress signals mediated by the T-cell receptor. All the cell lines expressed on their surface the high-affinity IL-2R (alpha, beta, and gamma c chains). They showed basal, constitutive phosphorylation, and coassociation of Jak3, STAT5, and STAT3. Weak basal phosphorylation of IL-2R gamma c was also detected. In regard to SzS, peripheral blood mononuclear cells from 10 of 14 patients showed basal phosphorylation of Jak3, accompanied by phosphorylation of STAT5 in 9 patients, and STAT3 in 4 patients. However, in vitro overnight culture of SzS cells without exogenous cytokines resulted in markedly decreased Jak3 and STAT5 phosphorylation, which could be reversed by stimulation with IL-2. This indicates that the basal phosphorylation of Jak3 and STAT5 in freshly isolated SzS cells is induced rather than constitutive. The basal activation of the Jak/STAT pathway involved in IL-2R signal transduction in ALCL and SzS cells reported here suggests that this pathway may play a role in the pathogenesis of cutaneous T-cell lymphomas, although the mechanism (induced versus constitutive) may vary between different lymphoma types.

Isolation of the human PC6 gene encoding the putative host protease for HIV-1 gp160 processing in CD4+ T lymphocytes.

Production of infectious HIV-1 virions is dependent on the processing of envelope glycoprotein gp160 by a host cell protease. The protease in human CD4+ T lymphocytes has not been unequivocally identified, yet members of the family of mammalian subtilisin-like protein convertases (SPCs), which are soluble or membrane-bound proteases of the secretory pathway, best fulfill the criteria. These proteases are required for proprotein maturation and cleave at paired basic amino acid motifs in numerous cellular and viral glycoprotein precursors, both in vivo and in vitro. To identify the gp160 processing protease, we have used reverse transcription-PCR and Northern blot analyses to ascertain the spectrum of SPC proteases in human CD4+ T cells. We have cloned novel members of the SPC family, known as the human PC6 genes. Two isoforms of the hPC6 protease are expressed in human T cells, hPC6A and the larger hPC6B. The patterns of SPC gene expression in human T cells has been compared with the furin-defective LoVo cell line, both of which are competent in the production of infectious HIV virions. This comparison led to the conclusion that the hPC6 gene products are the most likely candidates for the host cell protease responsible for HIV-1 gp160 processing in human CD4+ T cells.

Expression of a candidate cadherin in T lymphocytes.

Cadherins are homotypic adhesion molecules that classically mediate interactions between cells of the same type in solid tissues. In addition, E-cadherin is able to support homotypic adhesion of epidermal Langerhans cells to keratinocytes (Tang, A., Amagai, M., Granger, L. G., Stanley, J. R. & Udey, M. C. (1993) Nature (London) 361, 82-85) and heterotypic adhesion of mucosal epithelial cells to E-cadherin-negative intestinal intraepithelial T lymphocytes. Thus, we hypothesized that cadherins may play a wider role in cell-to-cell adhesion events involving T lymphocytes. We searched for a cadherin or cadherins in T lymphocytes with a pan-cadherin antiserum and antisera against alpha- or beta-catenin, molecules known to associate with the cytoplasmic domain of cadherins. The anti-beta-catenin antisera coimmunoprecipitated a radiolabeled species in T-lymphocyte lines that had a molecular mass of 129 kDa and was specifically immunoblotted with the pan-cadherin antiserum. Also, the pan-cadherin antiserum directly immunoprecipitated a 129-kDa radiolabeled species from an 125I surface-labeled Jurkat human T-cell leukemic cell line. After V8 protease digestion, the peptide map of this pan-cadherin-immunoprecipitated, 129-kDa species exactly matched that of the 129-kDa species coimmunoprecipitated with the beta-catenin antiserum. These results demonstrate that T lymphocytes express a catenin-associated protein that appears to be a member of the cadherin superfamily and may contribute to T cell-mediated immune surveillance.

The association between glycosylphosphatidylinositol-anchored proteins and heterotrimeric G protein alpha subunits in lymphocytes.

Glycosylphosphatidylinositol (GPI)-anchored proteins are nonmembrane spanning cell surface proteins that have been demonstrated to be signal transduction molecules. Because these proteins do not extend into the cytoplasm, the mechanism by which cross-linking of these molecules leads to intracellular signal transduction events is obscure. Previous analysis has indicated that these proteins are associated with src family member tyrosine kinases; however, the role this interaction plays in the generation of intracellular signals is not clear. Here we show that GPI-anchored proteins are associated with alpha subunits of heterotrimeric GTP binding proteins (G proteins) in both human and murine lymphocytes. When the GPI-anchored proteins CD59, CD48, and Thy-1 were immunoprecipitated from various cell lines or freshly isolated lymphocytes, all were found to be associated with a 41-kDa phosphoprotein that we have identified, by using specific antisera, as a mixture of tyrosine phosphorylated G protein alpha subunits: a small amount of Gialpha1, and substantial amounts of Gialpha2 and Gialpha3. GTP binding assays performed with immunoprecipitations of CD59 indicated that there was GTP-binding activity associated with this molecule. Thus, we have shown by both immunochemical and functional criteria that GPI-anchored proteins are physically associated with G proteins. These experiments suggest a potential role of G proteins in the transduction of signals generated by GPI-anchored molecules expressed on lymphocytes of both mouse and human.

cAMP inducibility of transcriptional repressor ICER in developing and mature human T lymphocytes.

Stimulation of the cAMP-dependent signaling pathway exerts an inhibitory effect on the proliferation and effector functions of T cells. The ability of T cells to form high intracellular levels of cAMP is acquired during development in the human thymus and is retained by the majority of mature peripheral T lymphocytes. Here we show that elevated cAMP levels in T cells correlate with the expression of the potent transcriptional repressor ICER (inducible cAMP early repressor) previously described in the hypothalamic-pituitary-gonadal axis. Further, in transcriptional assays in vivo, ICER inhibits calcineurin-mediated expression of the interleukin 2 promoter as well as Tax-mediated transactivation of the human T-lymphotropic virus type I (HTLV-I) promoter. Thus, the induction of ICER in T cells may play an important role in the cAMP-induced quiescence and the persistent latency of HTLV-I.

Hypothesis: "Rogue cell"-type chromosomal damage in lymphocytes is associated with infection with the JC human polyoma virus and has implications for oncopenesis.

The hemagglutination inhibition antibody titers against the JC and BK polyoma viruses (JCV and BKV, respectively) are significantly elevated in individuals exhibiting "rogue" cells among their cultured lymphocytes. However, the elevation is so much greater with respect to JCV that the BKV elevation could readily be explained by cross reactivity to the capsid protein of these two closely related viruses. The JCV exhibits high sequence homology with the simian papovavirus, simian virus 40 (SV40), and inoculation of human fetal brain cells with JCV produces polyploidy and chromosomal damage very similar to that produced by SV40. We suggest, by analogy with the effects of SV40, that these changes are due to the action of the viral large tumor antigen, a pluripotent DNA binding protein that acts in both transcription and replication. The implications of these findings for oncogenesis are briefly discussed.

CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma.

Bacterial infection stimulates the host to mount a rapid inflammatory response. A 6-base DNA motif consisting of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines was shown to contribute to this response by inducing polygonal B-cell activation. This stimulatory motif is 20 times more common in the DNA of bacteria than higher vertebrates. The current work shows that the same motif induces the rapid and coordinated secretion of interleukin (IL) 6, IL-12, and interferon gamma (but not IL-2, IL-3, IL-4, IL-5, or IL-10) in vivo and in vitro. Stimulatory CpG DNA motifs induced B, T, and natural killer cells to secrete cytokine more effectively than did lipopolysaccharide. Thus, immune recognition of bacterial DNA may contribute to the cytokine, as well as the antibody production characteristic of an innate inflammatory response.

Expression of a single-chain HLA class I molecule in a human cell line: presentation of exogenous peptide and processed antigen to cytotoxic T lymphocytes.

We have synthesized a recombinant gene encoding a single-chain HLA-A2/beta 2-microglobulin (beta 2m) molecule by linking beta 2m through its carboxyl terminus via a short peptide spacer to HLA-A2 (A*0201). This gene has been expressed in the beta 2m-deficient colorectal tumor cell line DLD-1. Transfection of this cell with the single-chain construct was associated with conformationally correct cell surface expression of a class I molecule of appropriate molecular mass. The single-chain HLA class I molecule presented either exogenously added peptide or (after interferon-gamma treatment) endogenously processed antigen to an influenza A matrix-specific, HLA-A2-restricted cytotoxic T-lymphocyte line. The need for interferon gamma for the processing and presentation of endogenous antigen suggests that DLD-1 has an antigen-processing defect that can be up-regulated, a feature that may be found in other carcinomas. Our data indicate that single-chain HLA class I constructs can form functional class I molecules capable of presenting endogenously processed antigens. Such molecules should be of use for functional studies, as well as providing potential anticancer immunotherapeutic agents or vaccines.

Induction of the WAF1/CIP1 protein and apoptosis in human T-cell leukemia virus type I-transformed lymphocytes after treatment with adriamycin by using a p53-independent pathway.

The WAF1/CIP1 protein has been identified as a downstream mediator of the tumor suppressor p53 in regulating cell cycle progression through a G1-phase check-point. Recent work has implicated the functional status of p53 as a critical determinant in the apoptotic response of certain cell lines to DNA damaging agents. By using human T-cell leukemia virus type I-transformed lymphoid cell lines that differ in their level and function of wild-type p53, we investigated the induction of WAF1/CIP1 and apoptosis after exposure to Adriamycin, a genotoxic agent. We found that regardless of the p53 status in these cell lines, WAF1/CIP1 RNA was rapidly induced in response to Adriamycin treatment. An elevated level of WAF1/CIP1 protein was observed as well. Additionally, we demonstrated that apoptosis was induced in all cell lines analyzed despite some having functionally inactive p53 protein. Our data suggest that a p53-independent pathway may play a role in the apoptotic response observed in some cell lines after exposure to DNA damaging agents.