The phosphatidylinositol response is an early event in the physiologically relevant activation of antigen-specific B lymphocytes

Receptor ligand-induced turnover of plasma membrane phosphatidylinositol (PI) has been implicated as part of a membrane receptor signal transduction system in a number of mammalian cell types. Signaling through B-lymphocyte surface immunoglobulin (sIg2) has been explored polyclonally through the use of anti-Ig reagents, with the assumption that anti-Ig mimics the process of antigen binding to the antigen-specific cell. We have utilized a method of obtaining trinitrophenyl (TNP)-specific populations of B lymphocytes in order to determine if antigen binding to these antigen-specific cells initiates PI turnover. This method has allowed us to explore the membrane phospholipid events following antigen binding directly, rather than with inference from the anti-Ig system. We have found that both thymus-dependent and thymus-independent antigens (with the exception of TNP-lipopolysaccharide) produced an increase in PI turnover comparable to that generated by anti-IgM stimulation. The lack of increased PI turnover following TNP-LPS stimulation may be attributable to the action of LPS on the biochemical events of the PI cycle. In a B-cell subpopulation depleted of antigen-specific cells, only anti-IgM produced a PI effect. These results represent the first demonstration of PI turnover as an early activation event in a physiologically relevant lymphocyte system.     

Elevation in phosphatidylethanolamine is an early but not essential event for cardiac cell differentiation

The biosynthesis of phosphatidylethanolamine was examined during differentiation of P19 teratocarcinoma cells into cardiac myocytes. P19 cells were induced to undergo differentiation into cardiac myocytes by the addition of dimethyl sulfoxide to the medium. Immunofluorescence labeling confirmed the expression of striated myosin 10 days postinduction of differentiation. The content of phosphatidylethanolamine increased significantly within the first 2 days of differentiation. [1,3-(3)H]Glycerol incorporation into phosphatidylethanolamine was increased 7.2-fold during differentiation, indicating an elevation in de novo synthesis from 1, 2-diacyl-sn-glycerol. The mechanism for the increase in phosphatidylethanolamine levels during cardiac cell differentiation was a 2.8-fold increase in the activity of ethanolaminephosphotransferase, the 1,2-diacyl-sn-glycerol utilizing reaction of the cytidine 5'-diphosphate-ethanolamine pathway of phosphatidylethanolamine biosynthesis. Incubation of P19 cells with the phosphatidylethanolamine biosynthesis inhibitor 8-(4-chlorophenylthio)-cAMP inhibited the differentiation-induced elevation in phosphatidylethanolamine levels but did not affect the expression of striated myosin. The results suggest that elevation in phosphatidylethanolamine is an early event of P19 cell differentiation into cardiac myocytes, but is not essential for differentiation to proceed.     

Tyrosine phosphorylation is an obligatory event in IL-2 secretion.

Activation of T lymphocytes leads to the production of the T cell growth factor IL-2 that regulates T cell proliferation. This activation is associated with several potential intracellular signalling events including increased activity of phospholipase C (PLC) and resultant increases in production of inositol phosphates and diacylglycerols. In addition, phosphorylation of specific intracellular proteins on serine, threonine, and tyrosine residues increases. The role of each of these events in IL-2 production is unclear. Using Western blotting with antiphosphotyrosine antibodies, we demonstrate that activation of murine T cells with mitogenic lectins or anti-CD3 antibodies leads to a rapid increase in tyrosine phosphorylation of proteins of 120, 72, 62, 55, and 40 kDa. Similar patterns of antiphosphotyrosine antibodies reactivity were observed in splenocytes, a T cell hybridoma, and a T lymphoma. Tyrosine phosphorylation was detectable within minutes of addition of mitogenic lectins and persisted for at least 6 h. Pretreatment of the cells with pertussis toxin did not inhibit tyrosine phosphorylation indicating that a pertussis toxin-sensitive G protein is not involved in signal transduction. Neither increasing cytosolic-free calcium nor activating protein kinase C mimicked the effects of mitogenic lectins suggesting that tyrosine phosphorylation was not a consequence of activation of PLC. This was confirmed by demonstrating that mitogenic lectins induced similar patterns of tyrosine phosphorylation in cells in which activation of the TCR leads to increased PLC activity and in cells in which PLC is not stimulated. To test whether tyrosine phosphorylation is linked to IL-2 secretion, we determined the effect of three specific tyrosine kinase inhibitors (tyrphostins) on tyrosine phosphorylation, IL-2 secretion, and cellular proliferation. The concentration dependence of inhibition of tyrosine phosphorylation and IL-2 production were similar. However, higher concentrations of the tyrphostins were required to inhibit constitutive proliferation of the T cell line indicating that inhibition of IL-2 secretion was not secondary to nonspecific toxic effects of the tyrphostins. Addition of the tyrphostins after mitogenic lectin decreased the amount of tyrosine phosphorylation and IL-2 secretion in parallel. This indicates that both tyrosine kinases and phosphatases are activated and that continuous tyrosine phosphorylation is likely required for IL-2 secretion. Therefore, tyrosine phosphorylation appears to represent an obligatory event in the transmembrane signaling processes that lead to IL-2 secretion.     

Changes in phospholipid metabolism during B lymphocyte activation

We have examined phospholipid metabolism in murine B lymphocytes stimulated with anti-Ig bound to Sepharose. T cell-depleted splenic B lymphocytes cultured with Sepharose-coupled, affinity-purified goat anti-mouse Ig (GAMIg) increased the incorporation of 32PO4 into phosphatidic acid and phosphatidylinositol within 3 hr and increased [3H]-thymidine uptake at 48 hr. No increase in labeling was observed in phosphatidylethanolamine, phosphatidylcholine, or phosphatidylserine. Based on both negative and positive selection procedures, it was demonstrated that these responses occurred in B lymphocytes. In contrast to the thymidine uptake response of the GAMIg-stimulated B lymphocytes, the phospholipid response did not require the presence of accessory cells or exogenous cytokines. The same selective changes in phospholipid metabolism were observed in neoplastic B lymphocytes (BCL1) after treatment with Sepharose anti-mu, but not with Sepharose anti-Ia or Sepharose normal Ig. The dose-response relationships of 32PO4 incorporation into phosphatidic acid and phosphatidylinositol and [3H] thymidine uptake were nearly identical in BCL1 cells. The results of these experiments indicate that interaction of B lymphocytes with insolubilized anti-Ig results in prompt and selective changes in phospholipid metabolism that appear to be correlated with B lymphocyte proliferation.     

Protein kinase C activation in B cells by indolactam inhibits anti-Ig-mediated phosphatidylinositol bisphosphate hydrolysis but not B cell proliferation

In order to examine the role of phosphatidylinositol bisphosphate (PIP2) hydrolysis in B cell activation, we studied the effect of various classes of protein kinase C (PKC) activators on anti-Ig-mediated B cell stimulation. Anti-Ig-stimulated PIP2 hydrolysis, elevations in [Ca2+]i, and induction of DNA synthesis were inhibited by PMA (a phorbol ester) as previously reported. In contrast, indolactam (an alkaloid PKC activator) inhibited PIP2 hydrolysis and elevations in [Ca2+]i, but stimulated rather than inhibited cellular proliferation. In order to examine whether the binding avidity of the PKC activators to PKC played a role in determining their activity to stimulate or inhibit B cell activation, we studied two other PKC activators, bryostatin and mezerein. Again, both inhibited anti-Ig mediated PIP2 hydrolysis and elevations in [Ca2+]i, whereas only the former inhibited induction of DNA synthesis. These data suggest that a) high levels of PIP2 hydrolysis and elevations in [Ca2+]i are not essential for anti-Ig-mediated induction of B cell DNA synthesis and b) activation of PKC may induce both stimulatory and inhibitory pathways of B cell activation, and whether stimulation or inhibition of cell activation is observed may depend on the combined intensity of these two signals.     

Perinuclear localization of cytosolic phospholipase A(2)alpha is important but not obligatory for coupling with cyclooxygenases

In response to Ca(2+) signaling, cytosolic phospholipase A(2)alpha (cPLA(2)alpha) translocates from the cytosol to the perinuclear membrane, where downstream eicosanoid-synthetic enzymes, such as cyclooxygenase (COX), are localized. Although the spatiotemporal perinuclear colocalization of cPLA(2)alpha and COXs has been proposed to be critical for their functional coupling leading to prostanoid production, definitive evidence for this paradigm has remained elusive. To circumstantiate this issue, we took advantage of a chimeric cPLA(2)alpha mutant harboring the C2 domain of protein kinase Calpha, which translocates to the plasma membrane following cell activation. Transfection analyses of the native or chimeric cPLA(2)alpha in combination with COX-1 or COX-2 revealed that, even though the arachidonate-releasing capacities of native and mutant cPLA(2)alpha were comparable, prostaglandin production by mutant cPLA(2)alpha was markedly impaired as compared with that by native cPLA(2)alpha. We thus conclude that the perinuclear localization of cPLA(2)alpha is preferential, even if not obligatory, for efficient coupling with COXs.     

JNK2 is required for efficient T-cell activation and apoptosis but not for normal lymphocyte development

BACKGROUND: The Jun N-terminal kinase (JNK) signaling pathway has been implicated in cell proliferation and apoptosis, but its function seems to depend on the cell type and inducing signal. In T cells, JNK has been implicated in both antigen-induced activation and apoptosis. RESULTS: We generated mice lacking the JNK2 isozymes. The mutant mice were healthy and fertile but defective in peripheral T-cell activation induced by antibody to the CD3 component of the T-cell receptor (TCR) complex - proliferation and production of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) were reduced. The proliferation defect was restored by exogenous IL-2. B-cell activation was normal in the absence of JNK2. Activation-induced peripheral T-cell apoptosis was comparable between mutant and wild-type mice, but immature (CD4(+) CD8(+)) thymocytes lacking JNK2 were resistant to apoptosis induced by administration of anti-CD3 antibody in vivo. The lack of JNK2 also resulted in partial resistance of thymocytes to anti-CD3 antibody in vitro, but had little or no effect on apoptosis induced by anti-Fas antibody, dexamethasone or ultraviolet-C (UVC) radiation. CONCLUSIONS: JNK2 is essential for efficient activation of peripheral T cells but not B cells. Peripheral T-cell activation is probably required indirectly for induction of thymocyte apoptosis resulting from administration of anti-CD3 antibody in vivo. JNK2 functions in a cell-type-specific and stimulus-dependent manner, being required for apoptosis of immature thymocytes induced by anti-CD3 antibody but not for apoptosis induced by anti-Fas antibody, UVC or dexamethasone. JNK2 is not required for activation-induced cell death of mature T cells.     

Activation of zeaxanthin is an obligatory event in the regulation of photosynthetic light harvesting.

By dynamic changes in protein structure and function, the photosynthetic membranes of plants are able to regulate the partitioning of absorbed light energy between utilization in photosynthesis and photoprotective non-radiative dissipation of the excess energy. This process is controlled by features of the intact membrane, the transmembrane pH gradient, the organization of the photosystem II antenna proteins and the reversible binding of a specific carotenoid, zeaxanthin. Resonance Raman spectroscopy has been applied for the first time to wild type and mutant Arabidopsis leaves and to intact thylakoid membranes to investigate the nature of the absorption changes obligatorily associated with the energy dissipation process. The observed changes in the carotenoid Resonance Raman spectrum proved that zeaxanthin was involved and indicated a dramatic change in zeaxanthin environment that specifically alters the pigment configuration and red-shifts the absorption spectrum. This activation of zeaxanthin is a key event in the regulation of light harvesting.     

Increased apoptosis in lamina propria B cells during polymicrobial sepsis is FasL but not endotoxin mediated

Recent studies from our laboratory demonstrated that mucosal lymphoid tissue such as Peyer's patch cells and lamina propria (LP) B lymphocytes from mice shows evidence of increased apoptosis after sepsis that is associated with localized inflammation/activation. The mechanism for this is poorly understood. Endotoxin as well as Fas/Fas ligand (FasL) have been shown to augment lymphocyte apoptosis; however, their contribution to the increase of apoptosis in LP B-cells during sepsis is not known. To study this, sepsis was induced by cecal ligation and puncture (CLP) in endotoxin-tolerant C3H/HeJ or FasL-deficient C3H/HeJ-FasL(gld) (FasL(-)) mice and LP lymphocytes were isolated 24 h later. Phenotypic, apoptotic, and functional indexes were assessed. The number of LP B cells decreased markedly in C3H/HeJ mice but not in FasL-deficient animals at 24 h after CLP. This was associated with comparable alteration in apoptosis and Fas antigen expression in the B cells of these mice. Septic LP lymphocytes also showed increased IgA production, which was absent in the FasL-deficient CLP mice. Furthermore, Fas ligand deficiency appeared to improve survival of septic challenge. These data suggest that the increase in B cell apoptosis in septic animals is partially due to a Fas/FasL-mediated process but not endotoxin.     

Glutamate availability is important in intramuscular amino acid metabolism and TCA cycle intermediates but does not affect peak oxidative metabolism.

Muscle glutamate is central to reactions producing 2-oxoglutarate, a tricarboxylic acid (TCA) cycle intermediate that essentially expands the TCA cycle intermediate pool during exercise. Paradoxically, muscle glutamate drops approximately 40-80% with the onset of exercise and 2-oxoglutarate declines in early exercise. To investigate the physiological relationship between glutamate, oxidative metabolism, and TCA cycle intermediates (i.e., fumarate, malate, 2-oxoglutarate), healthy subjects trained (T) the quadriceps of one thigh on the single-legged knee extensor ergometer (1 h/day at 70% maximum workload for 5 days/wk), while their contralateral quadriceps remained untrained (UT). After 5 wk of training, peak oxygen consumption (VO2peak) in the T thigh was greater than that in the UT thigh (P<0.05); VO2peak was not different between the T and UT thighs with glutamate infusion. Peak exercise under control conditions revealed a greater glutamate uptake in the T thigh compared with rest (7.3+/-3.7 vs. 1.0+/-0.1 micromol.min(-1).kg wet wt(-1), P<0.05) without increase in TCA cycle intermediates. In the UT thigh, peak exercise (vs. rest) induced an increase in fumarate (0.33+/-0.07 vs. 0.02+/-0.01 mmol/kg dry wt (dw), P<0.05) and malate (2.2+/-0.4 vs. 0.5+/-0.03 mmol/kg dw, P<0.05) and a decrease in 2-oxoglutarate (12.2+/-1.6 vs. 32.4+/-6.8 micromol/kg dw, P<0.05). Overall, glutamate infusion increased arterial glutamate (P<0.05) and maintained this increase. Glutamate infusion coincided with elevated fumarate and malate (P<0.05) and decreased 2-oxoglutarate (P<0.05) at peak exercise relative to rest in the T thigh; there were no further changes in the UT thigh. Although glutamate may have a role in the expansion of the TCA cycle, glutamate and TCA cycle intermediates do not directly affect VO2peak in either trained or untrained muscle.     

Increased endogenous retroviral gene expression is a consequence of lymphocyte activation

Plasma cells of line 151(5) chickens have been shown to express elevated levels of endogenous retroviral envelope glycoprotein (VEG), measured relative to levels expressed by both immature B cells and resting peripheral B lymphocytes. In this study we analyzed the relationship between peripheral blood lymphocyte (PBL) maturation and the level of VEG expression. A culture system was developed that would support maturation of pokeweed mitogen-activated peripheral B lymphocytes. As analyzed by cytofluorometry, both Ig+ and Ig- lymphoblasts present in the pokeweed mitogen-stimulated cultures expressed detectable levels of VEG in contrast to bursacytes and PBL. Similarly, Ig- blasts, which were present in concanavalin A-stimulated cultures of PBL and presumed to represent activated T cells, were also positive for the expression of VEG. Immature T cells, i.e., thymocytes, although negative by immunofluorescence analysis, expressed VEG at levels that were detectable by radioimmunochemical techniques. These results indicate that T cells as well as B cells constitutively express VEG, and that mitogenic activation of the resting lymphocyte induces an increase in VEG expression.     

Cutting edge: SDS-stable Fas microaggregates: an early event of Fas activation occurring with agonistic anti-Fas antibody but not with Fas ligand

The 45 kDa Fas or CD95 receptor triggers apoptosis via the caspase cascade when stimulated by its ligand FasL or by agonistic Abs. Activated Fas receptors seem to oligomerize very early into SDS-stable and reducing agent-resistant microaggregates of 200-250 kDa on SDS-PAGE. However, these microaggregates have so far only been reported using agonistic anti-Fas Abs, and no results have been reported using FasL. Here, we demonstrate that the microaggregates do not form in response to FasL, while they always appear in response to the agonistic Ab, in four different cell lines and in normal lymphocytes from human blood. Therefore, the Fas microaggregates are not required for the induction of apoptosis via FasL. These results also suggest that subtle differences exist in the apoptotic pathways triggered by anti-Fas agonistic Abs and by FasL.     

Cell-associated IgM, but not IgD or I-A/E, is important in the activation of suppressor T cells by antigen-primed B cells

The ability of transferred antigen-primed immune B cells to induce T cell-mediated suppression of the antibody response to Type III pneumococcal polysaccharide (SSS-III) could be blocked or eliminated by prior treatment of B cells with F(ab')2 anti-Ig or anti-IgM antibodies; however, F(ab')2 anti-IgD antibodies, or M5/114 (monoclonal anti-I-A/E antibody), had no effect on activation of suppression by SSS-III-primed B cells. Thus, cell-associated IgM antibody plays an important role in the activation of suppressor T cells during the antibody response to SSS-III.     

Mitotic recombination is an important mutational event following oxidative damage

The mutagenic effects of hydrogen peroxide (H(2)O(2)), a source of reactive oxygen species (ROS) have been determined in human lymphocytes. T-lymphocytes mutated at the autosomal HLA-A locus on chromosome 6 have been clonally isolated (N = 2097 clones) and the molecular basis of each clonal mutation characterised as due to intragenic, deletion or mitotic recombination mutation. H(2)O(2) caused a dose dependent increase in mutation frequency. There was no significant increase in the frequency of intragenic mutations. Mitotic recombination (MR) was responsible for 87% of the increase in mutation frequency induced by H(2)O(2) and gene deletion was responsible for 13%. MR results in loss of heterozygosity (LOH) distal to the recombination site. It is known that LOH is important in the initiation and progression of cancer. These results suggest that the biologically important consequence of some ROS may be LOH as a by-product of recombination repair. They also suggest that if our observations apply to ROS generally, then many of the mutations which accumulate with ageing or which are observed in cancer may be due to factors other than ROS.     

Early events in B-cell activation: anti-Lyb2, but not BSF-1, induces a phosphatidylinositol response in murine B cells

Previous studies have indicated that the murine surface antigen Lyb2 is involved in an activation pathway that apparently does not involve the surface immunoglobulin receptor. As sIg has been shown to transduce its activation signal through the breakdown of phosphatidylinositol (PI), and since activation via Lyb2 does not involve sIg, it was of interest to determine if binding to Lyb2 generates a PI response. We have demonstrated that an allele-specific monoclonal antibody to Lyb2 (anti-Lyb2 mab), which has previously been shown to drive B cells into S, also activated PI metabolism in these cells. This activation occurred in a dose-dependent and allele-specific manner. Antibodies to other B-cell surface molecules such as Ia did not induce a PI response. The effect of anti-Lyb2 mab was always less in magnitude than that induced by anti-IgM, but the effects of the two antibody preparations were most comparable in larger, presumptively preactivated cells. To explore the issue that Lyb2 may represent a receptor for a growth factor, possibly the early-acting B-cell growth factor BSF-1, we studied the PI response to BSF-1 and the effect of BSF-1 on Lyb2-induced PI turnover. BSF-1 neither induced a PI response nor inhibited competitively the response induced by anti-Lyb2 mab.     

Stat5 is essential for early B cell development but not for B cell maturation and function

The two closely related Stat5 (Stat5A and Stat5B) proteins are activated by a broad spectrum of cytokines. However, with the complication of the involvement of Stat5A/5B in stem cell function, the role of Stat5A/5B in the development and function of lymphocytes, especially B cells, is not fully understood. In this study, we demonstrated that Stat5A/5B(-/-) fetal liver cells had severe diminution of B cell progenitors but clearly had myeloid progenitors. Consistently, the mutant fetal liver cells could give rise to hemopoietic progenitors and myeloid cells but not B cells beyond pro-B cell progenitors in lethally irradiated wild-type or Jak3(-/-) mice. Deletion of Stat5A/5B in vitro directly impaired IL-7-mediated B cell expansion. Of note, reintroduction of Stat5A back into Stat5A/5B(-/-) fetal liver cells restored their abilities to develop B cells. Importantly, CD19-Cre-mediated deletion of Stat5A/5B in the B cell compartment specifically impaired early B cell development but not late B cell maturation. Moreover, the B cell-specific deletion of Stat5A/5B did not impair splenic B cell survival, proliferation, and Ig production. Taken together, these data demonstrate that Stat5A/5B directly control IL-7-mediated early B cell development but are not required for B cell maturation and Ig production.