T cell activation via the T cell receptor: a comparison between WT31 (defining alpha/beta TcR)-induced and anti-CD3-induced activation of human T lymphocytes.

The T cell receptor (TcR) heterodimer of alpha/beta glycoprotein is noncovalently associated with CD3 glycoprotein forming TcR/CD3 complex. The TcR have been shown to recognize antigen, and CD3 antigen is responsible for signal transduction. In this study we compared the effects of WT31 (defining alpha/beta TcR) monoclonal antibody (MoAb) and anti-CD3 MoAb on various steps of human T cell activation. Both antibodies depolarized plasma membranes, increased cell volume, induced IL-2 production and the expression of IL-2 receptors (CD25 antigen) and induced DNA synthesis. Furthermore, the two antibodies showed no synergistic effect on any of these parameters. However, both MoAb showed synergism with phorbol ester (PMA). WT31-induced T cell activation was Ca(2+)-dependent because the addition of EGTA to the medium inhibited DNA synthesis and CD25 antigen expression. The blockers of protein kinase C (PKC), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) and staurosporin, in a dose-dependent manner inhibited WT31-induced DNA synthesis. Cholera toxin but not the pertussis toxin inhibited WT31-induced T cell activation, suggesting involvement of G protein in WT31-induced T cell activation. These data indicate that WT31 antibody activates human T cells by a pathway that is similar to that of anti-CD3-induced T cell activation.     

Similar molecular requirements for antigen receptor-triggered secretion of interferon and granule enzymes by cytolytic T lymphocytes

At least two biologically significant responses are triggered by the crosslinking of the T-cell receptor (TcR) on the surface of cloned cytotoxic T lymphocytes (CTL): synthesis and secretion of macrophage-activating factor(s) (MAF) that can be attributed to interferon-gamma (IFN) and release of preformed cytolytic granules. We directly compared the molecular requirements for synthesis and secretion of IFN and secretion of granule enzymes triggered in the same cell by the same activating ligand (antigen or monoclonal antibody (mAb) to TcR). An increase in the surface density of activating ligand (immobilized anti-TcR mAb) enhanced both secretion of IFN and secretion of granules. Secretion of IFN occurred immediately after synthesis: only low (but detectable) levels of IFN were detected in cell cytosolic or particulate fractions isolated from Percoll gradients of lysed CTL, while very high levels of IFN were found in the stimulated CTL culture fluids. Inhibitors of RNA synthesis and protein synthesis blocked secretion of IFN, but did not inhibit release of preformed cytolytic granules. The requirement for TcR crosslinking in triggering both secretion of granules and secretion of IFN from CTL was pharmacologically reproduced by the synergistic action of PMA, a protein kinase C activator, and the Ca2+ ionophore A23187. Both secretion of IFN and secretion of granules were absolutely dependent upon extracellular Ca2+: EGTA completely blocked both TcR- and PMA/A23187-induced secretion of IFN and exocytosis of granules. These studies suggest that similar molecular mechanisms are involved in secretion of newly synthesized IFN and secretion of preformed cytolytic granules. One notable difference between the molecular requirements for the two secretory events was a much lower concentration requirement for PMA for IFN synthesis and secretion than for granule secretion in the synergistic interactions with A23187. Implications of these studies for the exocytosis model of cell-mediated cytotoxicity are discussed.     

Regulation of DNA metabolic enzymes upon induction of preB cell development and V(D)J recombination: up-regulation of DNA polymerase delta.

Withdrawal of interleukin-7 from cultured murine preB lymphocytes induces cell differentiation including V(D)J immunoglobulin gene rearrangements and cell cycle arrest. Advanced steps of the V(D)J recombination reaction involve processing of coding ends by several largely unidentified DNA metabolic enzymes. We have analyzed expression and activity of DNA polymerases alpha, beta, delta and epsilon, proliferating cell nuclear antigen (PCNA), topoisomerases I and II, terminal deoxynucleotidyl transferase (TdT) and DNA ligases I, III and IV upon induction of preB cell differentiation. Despite the immediate arrest of cell proliferation, DNA polymerase delta protein levels remained unchanged for approximately 2 days and its activity was up-regulated several-fold, while PCNA was continuously present. Activity of DNA polymerases alpha,beta and epsilon decreased. Expression and activity of DNA ligase I were drastically reduced, while those of DNA ligases III and IV remained virtually constant. No changes in DNA topoisomerases I or II expression and activity occurred and TdT expression was moderately increased early after induction. Our results render DNA polymerase delta a likely candidate acting in DNA synthesis related to V(D)J recombination in lymphocytes.     

Small interfering RNA and gene expression analysis using a multiplex branched DNA assay without RNA purification

The authors have developed a novel multiplex detection system that quantitatively measures the expression level of 11 messenger RNAs (mRNAs) directly from cell lysates or tissue homogenates without RNA purification. The system incorporates branched DNA (bDNA) technology from Bayer and a multiplex bead array platform from Luminex. In this study, a 21-nt synthetic small interfering RNA (siRNA; specifically designed to knockdown interleukin-8 [IL-8] expression) was delivered into HeLa cells. Using the multiplex bDNA assay, gene expression levels were measured simultaneously from cell lysates for 11 genes. After treating the HeLa cells for 20 h with phorbol myristate acetate (PMA), IL-8 mRNA levels were induced by almost 50-fold; transfection with 30 nM IL-8-specific siRNA reduced the PMA-induced IL-8 mRNA by 80%. In addition, PMA induced mRNA expression in IL-1alpha (3-fold) and IL-6 (4-fold); however, the IL-8 siRNA did not affect the expression of either of these 2 cytokine genes, indicating that the siRNA was selective for IL-8 mRNA expression. Three housekeeping genes' expression levels were measured under all conditions tested. The multiplex bDNA assay provides a powerful tool for quantitative multiplex gene expression analysis directly from cell lysates, which could be extremely valuable for conservation of rare or difficult-to-obtain samples.     

siRNA-mediated silencing of c-kit in mouse primary spermatogonial cells induces cell cycle arrest

Several genes/gene products are known to act in a concert to regulate the process of spermatogenesis. One such gene is c-kit, a transmembrane tyrosine kinase receptor which plays an indispensable role in the maturation and differentiation of spermatogonial germ cells (SGCs). In the present study, siRNA approach was used to assess the role of c-kit in survival and proliferation of murine primary SGCs. The effect of different concentrations of anti-c-kit siRNA-1 and siRNA-2 (0.15, 0.315, 0.625, 1.25, 2.50, 5, and 10 nM) on c-kit protein and mRNA expression at post-transfection time (0, 6, 12, 24, 48, and 72 hours) was assessed using an array of techniques such as flow cytometry, ELISA, Western blot, and RT-PCR. Transfection of cells with anti-c-kit siRNAs (0.15-10 nM) at various time points after (0-72 hours) showed significant knockdown c-kit mRNA and protein expression. MTT, Alamar blue assays, and RT-PCR were used to investigate the effects of c-kit silencing on survival, proliferation, distribution, and apoptosis of cells. Experiments were also conducted to determine the effects of c-kit knockdown on cell cycle distribution, DNA laddering, and apoptosis. The results indicated that the transfection with anti-c-kit siRNA induces DNA fragmentation and cell cycle arrest at G(2)/M phase leading to significant reduction in cell viability and proliferation. In addition, enhanced suppression of c-kit protein in P815 cells was observed after transfection as compared to ES-E14TG2alpha cells, suggesting early onset of c-kit protein repression in P815 cells leading to prolongation in cell doubling time. In conclusion, our data provide the first evidence of specific knockdown of c-kit expression in mouse primary SGCs, which emphasizes the critical role played by c-kit in germ cell survival, proliferation, and apoptosis.     

Biochemical events accompanying macrophage activation and the inhibition of colony-stimulating factor-1-induced macrophage proliferation by tumor necrosis factor-alpha, interferon-gamma, and lipopolysaccharide.

Agents that can arrest cellular proliferation are now providing insights into mechanisms of growth factor action and how this action may be controlled. It is shown here that the macrophage activating agents tumor necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma), and lipopolysaccharide (LPS) can maximally inhibit colony stimulating factor-1 (CSF-1)-induced, murine bone marrow-derived macrophage (BMM) DNA synthesis even when added 8-12 h after the growth factor, a period coinciding with the G1/S-phase border of the BMM cell cycle. This inhibition was independent of autocrine PGE2 production or increased cAMP levels. In order to compare the mode of action of these agents, their effects on a number of other BMM responses in the absence or presence of CSF-1 were examined. All three agents stimulated BMM protein synthesis; TNF alpha and LPS, but not IFN gamma, stimulated BMM Na+/H+ exchange and Na+,K(+)-ATPase activities, as well as c-fos mRNA levels. IFN gamma did not inhibit the CSF-1-induced Na+,K(+)-ATPase activity. TNF alpha and LPS inhibited both CSF-1-stimulated urokinase-type plasminogen activator (u-PA) mRNA levels and u-PA activity in BMM, whereas IFN gamma lowered only the u-PA activity. In contrast, LPS and IFN gamma, but not TNF alpha, inhibited CSF-1-induced BMM c-myc mRNA levels, the lack of effect of TNF alpha dissociating the inhibition of DNA synthesis and decreased c-myc mRNA expression for this cytokine. These results indicate that certain biochemical responses are common to both growth factors and inhibitors of BMM DNA synthesis and that TNF alpha, IFN gamma, and LPS, even though they all have a common action in suppressing DNA synthesis, activate multiple signaling pathways in BMM, only some of which overlap or converge.     

Effect of thapsigargin on cytoplasmic Ca2+ and proliferation of human lymphocytes in relation to AIDS

The tumor-promoting sesquiterpene lactone, thapsigargin, induced a dose-dependent increase of the cytoplasmic Ca2+ concentration ([ Ca2+]i) in human lymphocytes from a resting level between 100 and 150 nM up to about 1 microM. Half-maximum response was found at about 1 nM of thapsigargin, full response at 100 nM. The effect of thapsigargin on [Ca2+]i exceeded that of phytohaemagglutinin (PHA) which raised [Ca2+]i to maximum 300 nM. In combination with phorbol 12-myristate 13-acetate (PMA), thapsigargin stimulated the proliferation of normal lymphocytes to the same extent as did PHA, whereas the thapsigargin/PMA treatment could not restore the defective proliferation of AIDS lymphocytes in spite of the increased [Ca2+]i. Thapsigargin or PMA added separately had no stimulatory effects on cell proliferation. The thapsigargin/PMA treatment caused an increase in the interleukin-2 (IL-2) production of the lymphocytes, which was much higher than that caused by the PHA treatment, even in AIDS lymphocytes. Moreover, the thapsigargin/PMA treatment stimulated the expression of the IL-2 receptors on both normal and AIDS lymphocytes, similar to the effect of PHA. It is concluded that thapsigargin exerts its effects on lymphocyte proliferation by increasing [Ca2+]i, and that the general defect of AIDS lymphocytes, rather than being ascribed to the initiating signal systems, is associated with later events related to DNA synthesis and proliferation.     

Establishment and characterization of a clonal human T-cell line, MKB-1 derived from a patient with acute myeloblastic leukemia

A human T-cell line, designated as MKB-1, was established by cloning procedures in a suspension culture from a peripheral blood of a 17-year-old female patient with acute myeloblastic leukemia. The immunological marker profile of MKB-1 indicated that unlike a myeloid phenotype of the original leukemic cells, the cells were positive for CD3 (both cell surface and cytoplasm), T cell receptor (TcR) alpha/beta heterodimer, CD4, CD5, CD7, CD10, CD57 (Leu7), SN-1 and the cytoplasmic TcR beta chain. These findings indicate the T cell nature of the established cells. Terminal deoxynucleotidyl transferase (TdT) was also detected in 60%. We did not detect markers of human myeloid and B cell associated antigens, HLA-class II or immunoglobulin chains. Cytogenetic study revealed that the MKB-1 cells had a female hypo-tetraploid karyotype with chromosomal abnormalities including a translocation between chromosomes 10 and 14. The breakpoint of chromosome 14 of this translocation, 14q11.2, is known to be the location of TcR alpha and delta genes; t(10; 14) (q26; q11.2) is a variant type of a T cell neoplasm-associated translocation, t (10; 14) (q24; q11.2). The MKB-1 cell line is unusual in that its T cell characteristics are phenotypically and cytogenetically distinct from the original myeloid leukemia cells.     

Folate deficiency inhibits the proliferation of primary human CD8+ T lymphocytes in vitro

Folate is required for one-carbon transfer reactions and the formation of purines and pyrimidines for DNA and RNA synthesis. Deficiency of folate can lead to many clinical abnormalities, including macrocytic anemia, cardiovascular diseases, birth defects, and carcinogenesis. The nucleotide imbalance due to folate deficiency causes cell cycle arrest in the S phase and uracil misincorporation into DNA, which may result in DNA double-strand breaks during repair. The role of folate in the immune system has not been fully characterized. We cultured PHA-activated human T lymphocytes in varying concentrations of folate, and measured proliferation, cell cycle, apoptosis, uracil misincorporation, and proportions of Th cells (CD4(+)) and cytotoxic T (CD8(+)) cells. Folate deficiency reduced proliferation of T lymphocytes, induced cell cycle arrest in the S phase, induced apoptosis, and increased the level of uracil in DNA. Folate deficiency also increased the CD4(+) to CD8(+) ratio due to a marked reduction of CD8(+) cell proliferation. Folate or nucleoside repletion of folate-deficient cells rapidly restored T lymphocyte proliferation and normal cell cycle, reduced the DNA uracil content, and lowered the CD4(+) to CD8(+) ratio. These data suggest that folate status may affect the immune system by reducing the capacity of CD8(+) cells to proliferate in response to activation.     

Tumor-promoting phorbol esters stimulate the proliferation of interleukin-3 dependent cells

To determine whether activation of protein kinase C is involved in the proliferation of interleukin-3 (IL-3) -dependent cells, we examined the effect of tumor-promoting phorbol esters on the in vitro proliferation of the IL-3-dependent cell lines FD and DA-1. The viability of FD and DA-1 cells cultured for 24 hours in 100 nM phorbol myristate acetate (PMA) and 10% FCS was similar to that of cells cultured in 25% WEHI-3 conditioned medium as a source of IL-3, and 10% FCS. FD cells failed to proliferate in concentrations of FCS of up to 50%, while DA-1 cell proliferation was not markedly influenced by FCS. By contrast, PMA promoted the proliferation of FD and DA-1 cells in the absence of FCS and enhanced their proliferation in the presence of 10% FCS, 60- and 20-fold, respectively. Stimulation of proliferation was achieved with as little as 10 nM PMA and was maximal at 100 nM PMA. Low concentrations (0.05-0.1%) of WEHI-3 CM promoted the proliferative response of FD and DA-1 cells to PMA, but at concentrations of WEHI-3 CM greater than 0.8%, no further increment in proliferation was obtained with PMA. As little as 1/2 hour of exposure to phorbol esters was sufficient to cause translocation of protein kinase C from the cytosol to the membranes of DA-1 cells, and 1 hour of exposure to phorbol esters was sufficient to stimulate DNA synthesis. A protein kinase C inhibitor, H-7, at a concentration of 10 microM inhibited phorbol ester-induced stimulation of DA-1 cell proliferation. When DA-1 cells were exposed to the calcium ionophore A23187 in addition to both a phorbol ester and IL-3, their proliferation was enhanced over that stimulated by only the phorbol ester and IL-3. The data indicate that stimulation of proliferation of IL-3-dependent cells involves the activation of protein kinase C.     

Signals involved in T cell activation. II. Distinct roles of intact accessory cells, phorbol esters, and interleukin 1 in activation and cell cycle progression of resting T lymphocytes

The signals involved in the initiation of mitogen-induced activation of resting guinea pig T cells were examined. The combination of phytohemagglutinin (PHA) and 4 beta-phorbol 12-myristate 13-acetate (PMA) stimulated DNA synthesis by accessory cell (AC)-depleted T cells cultured at high density, but the use of low density cultures indicated that intact AC were absolutely necessary for PHA-stimulated T cell DNA synthesis even in the presence of PMA, interleukin 1 (IL 1), or interleukin 2 (IL 2). In contrast, AC-depleted T cells were able to respond to the combination of the calcium ionophore, ionomycin, and PMA regardless of the cell density at which they were cultured. Cell cycle analysis by acridine orange staining indicated that neither PHA nor ionomycin, in the absence of AC, activated resting T cells. PMA in the absence of all AC, supported cell cycle entry and progression to the DNA synthetic phase of the majority of ionomycin-stimulated T cells, but permitted only a small number of PHA-triggered T cells to enter the initial stage of the cell cycle (G1a) characterized by a modest increase in cellular RNA content. Although PMA permitted some PHA-stimulated T cells to enter the cell cycle, most required intact AC to enter G1, and all required intact AC to progress through G1 and synthesize maximal amounts of RNA. No PHA-stimulated cells reached the S phase without intact AC. In PHA-stimulated cultures containing intact AC, PMA increased the number of cells entering the cell cycle and increased the rate of their progress to the DNA synthetic phase. IL 1 also augmented PHA-stimulated AC-dependent T cell DNA synthesis in the presence or absence of PMA, but appeared to be most active during the later stage of the first cell cycle, augmenting the number of activated cells that entered the S phase of the cell cycle. These results support the conclusion that intact AC, IL 1, and a PMA-like signal play distinct roles in the progression of mitogen-stimulated T cells through the first round of the cell cycle.     

Nonequivalent effects of PKC activation by PMA on murine CD4 and CD8 cell-surface expression

The membrane glycoproteins CD4 (L3T4) and CD8 (Lyt2) are expressed on distinct populations of mature murine T lymphocytes, and are thought to be receptors for monomorphic determinants expressed on MHC class II and class I molecules, respectively. Although they differ in their ligand specificity, it has been presumed that CD4 and CD8 perform equivalent functions in the T cells that bear them. Since activation of protein kinase C (PKC) is known to cause rapid down-regulation of various receptors, including the T cell receptor complex (TcR complex), we treated cells with phorbol 12-myristate 13-acetate (PMA), a PKC activator, to determine whether cell-surface expression of CD4 and CD8 would be similarly affected by this intracellular mediator. Brief or relatively prolonged treatment with PMA induced mature murine T cells to reduce their surface expression of the TcR complex and of CD4, but not of CD8. Similarly, PMA rapidly induced transfected L cells to down-regulate surface CD4 expression, but had no effect on surface CD8 expression. Most significantly, PMA treatment induced CD4+CD8+ immature thymocytes to rapidly reduce their surface CD4 expression, but, again, it had no immediate effect on the surface expression of CD8. These results indicate that CD4 and TcR complex cell-surface expression are both sensitive to PKC activation by brief treatment with PMA, whereas CD8 expression is not, and suggest that CD4 and CD8 surface expression levels are regulated by distinct intracellular mechanisms.     

WS-1 human fibroblasts contain distinct calcium and protein kinase C-mediated pathways for activation of Na+/H+ exchange: contrasting effects of thrombin and PMA

PMA and thrombin were examined for their ability to activate Na+/H+ exchange in growth-arrested WS-1 human fibroblasts. PMA or thrombin caused a cytoplasmic alkalinization that required extracellular sodium and was sensitive to 1 mM amiloride, suggesting that the rise in pH was mediated by the Na+/H+ exchanger. However, PMA and thrombin activated Na+/H+ exchange by distinctly different mechanisms. The rate of cytoplasmic alkalinization caused by 30 nM PMA was slower than 10 nM thrombin. The PMA-induced pH change was sensitive to the protein kinase inhibitors staurosporine (50 nM) and H-7 (100 microM). No increase in intracellular calcium was observed after PMA treatment and the cytoplasmic alkalinization caused by PMA was not sensitive to the drug TMB8 (200 microM) or the intracellular calcium-chelator BAPTA. In contrast, the thrombin-induced rise in cytoplasmic pH was insensitive to 50 nM staurosporine and only partially reduced with 100 microM H-7. The thrombin-induced activation of Na+/H+ exchange was inhibited by 200 microM TMB8 or pretreatment with BAPTA. PMA caused translocation of PKC activity from a cytoplasmic to membrane fraction whereas thrombin did not. Pretreatment with 50 nM staurosporine significantly reduced measurable PKC activity with or without PMA treatment. PMA and thrombin were also examined for their ability to induce DNA synthesis in growth-arrested WS-1 human fibroblasts. Unlike thrombin, PMA did not stimulate [3H]-thymidine incorporation in cells serum-deprived for 48 hours. In addition, PMA inhibited thrombin-induced DNA synthesis when added at the same time or as late as 10 hours after thrombin addition. Therefore, thrombin and PMA activate Na+/H+ exchange by distinct pathways, but only the thrombin-induced pathway correlates with a mitogenic response.