Human germinal center B cells express the apoptosis-inducing genes Fas, c-myc, P53, and Bax but not the survival gene bcl-2.

During T cell-dependent antibody responses, B cells within germinal centers (GC) alter the affinity of their antigen receptor by introducing somatic mutations into variable region of immunoglobulin (IgV) genes. During this process, GC B cells are destined to die unless positively selected by antigens and CD40-ligand. To understand survival/death control of germinal center B cell, the expression of four apoptosis-inducing genes, Fas, c-myc, Bax, and P53, together with the survival gene bcl-2, has been analyzed herein among purified tonsillar naive, GC, and memory B cells. IgD+CD38- naive B cells were separated into CD23- (mature B cell [Bm]1) subset and CD23+ (Bm2), IgD-CD38+ GC B cells were separated into subsets of CD77+ centroblasts (Bm3) and CD77- centrocytes (Bm4), whereas IgD-CD38- cells represented the Bm5 memory B cell subset. Sequence analysis of IgV region genes indicated that somatic hypermutation was triggered in the Bm3 centroblast subset. Here we show that bcl-2 is only detectable with naive (Bm1 and 2) and memory B cell (Bm5) subsets, whereas all four apoptosis-inducing genes were most significantly expressed within GC B cells. Fas was equally expressed in Bm3 centroblasts and Bm4 centrocytes, whereas Bax was most significantly expressed in Bm4 centrocytes. c-myc, a positive regulator of cell cycle, was most significantly expressed in proliferating Bm3 centroblasts, whereas P53, a negative regulator of cell cycle, was most signficantly expressed in nonproliferating Bm4 centrocytes. The present results indicate that the survival/death of GC B cells are regulated by the up- and downregulation of multiple genes, among which the expression of c-myc and P53 in the absence of bcl-2 may prime the proliferating Bm3 centroblasts and nonproliferating Bm4 centrocytes to apoptosis.

Interleukin-10 induces immunoglobulin G isotype switch recombination in human CD40-activated naive B lymphocytes.

Upon activation, B lymphocytes can change the isotype of the antibody they express by immunoglobulin (Ig) isotype switch recombination. In previous studies on the regulation of human IgG expression, we demonstrated that interleukin 10 (IL-10) could stimulate IgG1 and IgG3 secretion by human CD40-activated naive (sIgD+) tonsillar B cells. To assess whether IL-10 actually promotes the DNA recombination underlying switching to these isotypes, we examined the effect of IL-10 on the generation of reciprocal products that form DNA circles as by-products of switch recombination. The content of reciprocal products characteristic of mu-gamma recombination was elevated after culture of CD40-activated tonsillar sIgD+ B cells with either IL-4 or IL-10, although high levels of IgG secretion were observed only with IL-10. Unlike IL-4, IL-10 did not induce reciprocal products of mu-epsilon and gamma-epsilon switch recombination. These results demonstrate that IL-10 promotes both switching to gamma and IgG secretion.

Depletion of CD8+ cells in human thymic medulla results in selective immune deficiency.

CD8 molecules expressed on the surface of a subset of T cells participate in the selection of class I MHC antigen-restricted T cells in the thymus, and in MHC-restricted immune responses of mature class I MHC antigen-restricted T cells. Here we describe an immune-deficient patient with lack of CD8+ peripheral blood cells. The patient presented with Pneumocystis carinii pneumonia and was unable to reject an allogeneic skin graft, but had normal primary and secondary antibody responses. Examination of the patient's thymus revealed that the loss of CD8+ cells occurred during intrathymic differentiation: the patient's immature cortical thymocytes included both CD4+ and CD8+ cells while the mature medullary cells expressed the CD4 but not the CD8 protein on their surface. Northern blot and polymerase chain reaction analyses revealed the presence of CD8 alpha and beta mRNA in the patient's thymus but not in the peripheral blood. Both class I MHC antigen expression and the expressed TCR V beta repertoire are normal in this patient. These data are consistent with an impaired selection of CD8+ cells in the patient's thymus and support the role of the CD8 surface protein in thymic selection previously characterized in genetically manipulated and inbred mice.

Interleukin 10 induces B lymphocytes from IgA-deficient patients to secrete IgA.

We have previously shown that human B lymphocytes cultured in the CD40 system, composed of an anti-CD40 mAb presented by a CD32-transfected fibroblastic cell line, proliferate but do not secrete antibodies. However, the addition of particles of Staphylococcus aureus Cowan (SAC) induces B cell differentiation even in the absence of exogenous cytokines (CD40/SAC system). Additionally, B lymphocytes cultured in the CD40 system in the presence of human IL-10, produce IgM, IgG, and IgA, and Ig levels are further increased by SAC. Here, we have studied the capacity of peripheral blood lymphocytes from patients with IgA deficiency (IgA-D) to secrete Igs, particularly IgA after CD40 triggering. Peripheral blood mononuclear cells (PBMNC) from IgA-D patients cultured in the CD40/SAC system produced IgM and IgG, but not IgA. The addition of IL-10 to the cultures, enhanced the production of IgM and IgG and most strikingly induced the production of high amounts of IgA. The addition of IL-10 to PBMNC from IgA-D patients activated through CD40 alone resulted in the production of IgA. Thus, SAC and anti-CD40 mAb stimulate B cells to differentiate into cells secreting IgG and IgM whereas IL-10 plays a central role in inducing B cells from IgA-D patients to differentiate into IgA secreting cells.

Phorbol esters induce changes in adenosine deaminase, purine nucleoside phosphorylase, and terminal deoxynucleotidyl transferase messenger RNA levels in human leukemic cell lines.

We have studied the expression of mRNA encoding adenosine deaminase (ADA; EC 3.5.4.4), purine nucleoside phosphorylase (PNP; EC 2.4.2.1), and terminal deoxynucleotidyltransferase (TdT; EC 2.7.7.31) in different leukemic cell lines of B- and T-cell lineage. Incubation of leukemic cells in the presence of the phorbol esters, 12-O-tetradecanoyl-phorbol-13-acetate or phorbol 12,13-dibutyrate, resulted in reduction of ADA and TdT mRNA levels, while PNP mRNA levels increased under the same treatment. The effect of TPA on the activity of these enzymes correlated well with its effects on their mRNA levels. TPA caused a 40% decrease in ADA and a 60% decrease in TdT enzyme activity, after 6 h of treatment. In contrast, PNP activity increased up to 200% after 12 h of incubation with the phorbol ester. The changes induced by the phorbol esters in the levels of mRNA of ADA, PNP, and TdT, and their enzyme activities in human leukemic cell lines mimic the changes in the activities of these enzymes in developing T-lymphocytes during differentiation in vivo, suggesting a role for protein kinase C in the regulation of ADA, PNP, and TdT gene expression during lymphoid cell differentiation.

Characterization of a t(1;19) pre-B acute lymphoblastic leukemia (ALL) cell line which proliferates in response to IL-7.

The present study describes the establishment of the cell line Pre-Alp from the bone marrow of a pediatric patient with a t(1;19) pre-B acute lymphoblastic leukemia (ALL) at diagnosis. Proliferation of leukemic blasts was found to be initially dependent on the presence of autologous stromal cells. However, after five weeks of culture, the stromal cells were no longer necessary and cells began to grow autonomously, with a doubling time of approximately 24 hours. The established Pre-Alp cell line displays a pre-B cell phenotype (CD19+, CD10+, CD34-, c mu+, s mu-), with immunoglobulin (Ig) light chain DNA in germline configuration, and carries a (1;19)(p23;q13.3) chromosomal translocation identical to the freshly-isolated leukemic blasts. A unique feature of this cell line is represented by its ability to respond to interleukin 7 (IL-7). Thus, IL-7 enhances 3H-thymidine uptake by Pre-Alp cells in a dose-dependent manner, under conditions of low cell density and serum concentration, and increases cell recovery. Finally, Pre-Alp cells were found to remain at a pre-B stage even upon addition of various cytokines, which failed to induce a transition to surface Ig+ cells. The presently described cell line should constitute a useful model of t(1;19) pre-B ALL and permit the study of IL-7 dependent signal transduction in human pre-B cells.

In vivo effect of adenosine on adenine nucleotides and inorganic phosphate in rat blood.

The effect of adenosine and the time response on adenine nucleotide and Pi levels in rat blood was investigated. An increase in adenine nucleotide with a concomitant decrease in Pi concentration 30, 60, and 90 minutes after the nucleoside administration were observed. Though the 100 mg/Kg dose showed the highest effect on nucleotide concentration, the maximal response on Pi content was achieved with the 50 mg/Kg dose. The results are discussed at the light of previous data obtained in hepatocytes, and using as indicators the energy charge and the phosphorylation potential.

Regulation of gamma T-cell antigen receptor expression by intracellular calcium in acute lymphoblastic leukemia cell line DND41.

The calcium ionophore, ionomycin, promotes an increase of intracellular calcium and regulates mRNA expression of gamma/delta-TcR gene in human T lymphocytes. The mechanism of this regulation is not yet clear. Thus, the regulation by intracellular calcium requires elucidation. We studied the gamma-TcR gene expression in acute lymphoblastic leukemia cell line DND41 (CD4- CD8-) by Northern blot and flow cytometric analysis. The mRNA levels of gamma-TcR increased by ionomycin, anti-CD3, and with TPA. TPA had an antagonistic effect to both ionomycin and anti-CD3. Also, TPA inhibits the increased intracellular calcium promoted by ionomycin but not the increase promoted by anti-CD3 and ionomycin. Our results suggest that intracellular calcium induces mRNA and protein expression of gamma-TcR chain. This effect is antagonized by protein kinase C-activation. Thus, we conclude that the target cells of the differential regulation on gamma-TcR mRNA expression by intracellular calcium modulators are the CD4- CD8- cells, and this is due to cytosolic calcium mobilization.

Vital function of PRELI and essential requirement of its LEA motif.

Proteins containing the late embryogenesis abundant (LEA) motif comprise a conserved family, postulated to act as cell protectors. However, their function and mechanisms of action remain unclear. Here we show that PRELI, a mammalian LEA-containing homolog of yeast Ups1p, can associate with dynamin-like GTPase Optic Atrophy-1 (OPA1) and contribute to the maintenance of mitochondrial morphology. Accordingly, PRELI can uphold mitochondrial membrane potential (ΔΨ(m)) and enhance respiratory chain (RC) function, shown by its capacity to induce complex-I/NADH dehydrogenase and ATP synthase expression, increase oxygen consumption and reduce reactive oxygen species (ROS) production. PRELI can also inhibit cell death induced by STS, TNF-α or UV irradiation. Moreover, in vitro and in vivo dominant-negative overexpression of mutant PRELI/LEA(-) (lacking the LEA motif) and transient in vitro PRELI-specific knockdown can render lymphocytes vulnerable to apoptosis, cause mouse embryo lethality and revert the resistance of lymphoma cells to induced death. Collectively, these data support the long-presumed notion of LEA protein-dependent mechanisms of cytoprotection and suggest that PRELI interacts with OPA1 to maintain mitochondria structures intact, sustain balanced ion(-)/proton(+) gradients, promote oxidative phosphorylation reactions, regulate pro- and antiapoptotic protein traffic and enable cell responses to induced death. These findings may help to understand how bioenergetics is mechanistically connected with cell survival cues.

Coordinate regulation of mRNAs encoding adenosine deaminase, purine nucleoside phosphorylase, and terminal deoxynucleotidyltransferase by phorbol esters in human thymocytes.

Incubation of human thymocytes in the presence of phorbol esters caused a reversible decrease in the mRNAs encoding terminal deoxynucleotidyltransferase (TdT; EC 2.7.7.31) and adenosine deaminase (ADA; EC 3.5.4.4) and an increase in the mRNA encoding purine nucleoside phosphorylase (PNP; EC 2.4.2.1). The effect of phorbol esters on TdT and ADA mRNA levels can be attributed to an apparent decrease in the stability of the mRNAs. The changes in ADA, TdT, and PNP mRNAs closely simulate changes in the activities of these enzymes that occur during T-cell differentiation in vivo, suggesting a role for protein kinase C activation in the regulation of the expression of these genes during intrathymic T-cell differentiation. A role for these purine degradation enzymes in the regulation of intracellular pools of the deoxynucleotide substrates of TdT is discussed.

Coordinate transcriptional regulation of alpha and delta chains of the T-cell antigen receptors by phorbol esters and cyclic adenosine 5'-monophosphate in human thymocytes.

The regulation of T-cell antigen receptor (TcR) gene expression by phorbol ester and cAMP was studied in human thymocytes. Incubation of human thymocytes in the presence of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate increased the levels of the mRNAs of TcR-alpha and -delta while the levels of TcR-beta and -gamma mRNA were not affected. cAMP, on the other hand, caused a decrease in TcR-alpha and -delta mRNA levels but also did not affect TcR-beta and -gamma mRNA levels. The induction of TcR-alpha and -delta mRNA by 12-O-tetradecanoylphorbol-13-acetate occurred at the transcriptional level only whereas cAMP treatment decreased both TcR-alpha gene transcription and the stability of its mRNA. The similarity in the transcriptional control of TcR-alpha and -delta genes combined with their close chromosomal location raises the possibility that they share common DNA regulatory elements.

Phorbol esters and calcium ionophores differentially regulate the transcription of gamma-T-cell antigen receptor gene in human thymocytes.

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and the calcium ionophore ionomycin differentially regulated the expression of the gamma-T-cell antigen receptor (gamma-TCR) gene in human thymocytes. Ionomycin induced an increase in gamma-TCR mRNA while TPA prevented this effect. The effects of ionomycin and TPA are completely reversed upon their removal. Protein kinase C-depleted cells are unable to respond to TPA but the induction of gamma-TCR by ionomycin was unimpaired. The regulation of gamma-TCR gene expression by TPA and ionomycin occurred at the level of gene transcription, whereas the rate of gamma-TCR mRNA degradation was unaffected by TPA or ionomycin.

Inhibition of the production of proinflammatory cytokines and immunoglobulins by interleukin-4 in an ex vivo model of rheumatoid synovitis.

OBJECTIVE: To assess the spontaneous production of proinflammatory cytokines and immunoglobulins in rheumatoid arthritis (RA) synovitis and modulation by interleukin-4 (IL-4). METHODS: We developed an ex vivo model of RA synovitis using pieces of RA synovium, and have studied the regulation of the production of IL-1 beta, IL-6, tumor necrosis factor alpha (TNF alpha), IgM, and IgG. RESULTS: Spontaneous production of proinflammatory cytokines in vitro was active, with prolonged cytokine gene transcription and translation. IL-6 was produced at higher levels than either IL-1 beta or TNF alpha, and explants produced more IgG than IgM. In contrast, IL-4 and interferon-gamma were undetectable. When pieces of synovium were incubated in the presence of IL-4, reduction of spontaneous proinflammatory cytokine and Ig production was observed. CONCLUSION: These results extend the observations of the antiinflammatory properties of IL-4 to an ex vivo situation, and provide the rationale for the clinical use of IL-4 in RA.

B-chronic lymphocytic leukemias can undergo isotype switching in vivo and can be induced to differentiate and switch in vitro.

B-chronic lymphocytic leukemias (B-CLL) represent expanded clones of resting B lymphocytes that mostly express surface IgM (sIgM). The present study shows that B-CLL cells, freshly isolated from two patients, were sIgM+, sIgG-, and sIgA- but expressed IgG and IgA transcripts. cDNA cloning and sequencing showed that the VDJ segments associated to gamma and alpha heavy chain transcripts are identical to those from mu transcripts, thus showing that B lymphocytes giving rise to CLL cells have undergone isotype switching in vivo. Stimulation of these B-CLL cells through surface CD40 in the presence of interleukin-10 induced them to secrete IgG and IgA, proving that they can also differentiate into Ig-secreting cells. Finally, CD40-stimulated B-CLL cells were induced to switch towards IgE in response to interleukin-4, as shown by the presence of specific VDJ-C epsilon transcripts and the secretion of IgE. Therefore, B-CLL cells tested herein can undergo isotype switching in vivo and can be induced to undergo further isotype switching and differentiation in vitro.

The expression of c-fos, c-jun, and c-myc genes is regulated by heat shock in human lymphoid cells.

The effect of heat shock on the expression of the nuclear protooncogenes c-fos, c-jun, and c-myc was studied in human lymphoid cells. Heat shock caused an increase in c-fos and c-jun mRNA levels and a decrease in c-myc mRNA levels in pre-B (Hyon) and T (DND-41) cell lines as well as in freshly isolated normal human thymocytes. The changes in the mRNA levels of these protooncogenes in Hyon cells were most pronounced at 42 and 43 degrees C; kinetic analysis demonstrated that the changes could be detected within 30 min of heat shock. Altered transcription of c-fos and c-myc genes was the primary effect of heat shock. Secondarily, heat shock of Hyon cells stabilized the c-myc mRNA level by increasing its half-life from 24 to 45 min. The overall effect of heat shock on c-myc mRNA level, however, was a marked inhibition of its transcription. These results demonstrate that the transcription of nuclear protooncogenes is regulated by heat shock indicating a role for nuclear protooncogenes in the stress response of lymphoid cells.

Differential regulation of gamma and delta T cell antigen receptor gene expression by phorbol esters and Ca2+ ionophores in the acute lymphocyte leukemia DND41 cell line.

We have investigated the role of two signal transduction pathways on the regulation of the gamma and delta T cell antigen receptor (TcR) gene expression, in the acute lymphocytic leukemic cell line DND41. Protein kinase C (PKC) activation, and intracellular free Ca2+ mobilization, initiated by phorbol esters and calcium ionophores, respectively, not only acted independently but, more interestingly, their effects were antagonistic, suggesting a role for these signals during T cell differentiation. The Ca2+ ionophore, ionomycin, increased the levels of intracellular free Ca2+ and induced the expression of the gamma and delta chains of the T cell antigen receptor in a concentration-dependent manner. The phorbol ester 12-myristate 13-acetate down-regulated the basal gamma TcR expression with marginal effect on delta TcR mRNA, but diminished the induction of both gamma and delta TcR, initiated by the Ca2+ ionophore. These antagonistic effects of the two arms of the phospholipase C-mediated signal transduction pathways, i.e. PKC activation and increased intracellular free Ca2+, were specific to the regulation of the gamma and delta TcR, since the same signals exerted a synergistic effect on the mRNA levels of interleukin 2 receptor. These data confirm our hypothesis that the antagonistic regulation on the gamma and delta TcR gene expression by phorbol esters and calcium ionophores occurs in the same cell, and stresses the biological significance of PKC activation and intracellular free calcium mobilization during intrathymic differentiation and selection.