B-lineage Cell Lines Research Articles

Liposome-encapsulated progesterone efficiently suppresses B-lineage cell proliferation

Progesterone suppresses several ancient pathways in a concentration-dependent manner. Based on these characteristics, progesterone is considered a candidate anticancer drug. However, the concentration of progesterone used for therapy should be higher than the physiological concentration, which makes it difficult to develop progesterone-based anticancer drugs. We previously developed liposome-encapsulated progesterone (Lipo-P4) with enhanced anticancer effects, which strongly suppressed triple-negative breast cancer cell proliferation in humanized mice. In this study, we aimed to clarify whether Lipo-P4 effectively suppresses the proliferation of B-lineage cancer cells. We selected six B-cell lymphoma and two myeloma cell lines, and analyzed their surface markers using flow cytometry. Next, we prepared liposome-encapsulated progesterone and examined its effect on cell proliferation in these B-lineage cancer cells, three ovarian clear cell carcinoma cell lines, two prostate carcinoma cell lines, and one triple-negative breast cancer adenocarcinoma cell line. Lipo-P4 suppressed the proliferation of all cancer cell lines. All B-lineage cell lines, except for the HT line, were more susceptible than the other cell types, regardless of the expression of differentiation markers. Empty liposomes did not suppress cell proliferation. These results suggest that progesterone encapsulated in liposomes efficiently inhibits the proliferation of B-lineage cells and may become an anticancer drug candidate for B-lineage cancers.

Targeting of interleukin-10 receptor by a potential human interleukin-10 peptide efficiently blocks interleukin-10 pathway-dependent cell proliferation.

Objective:Human interleukin-10 (IL-10) is a dimeric and pleiotropic cytokine that plays a crucial role in cellular immunoregulatory responses. As IL-10 binds to its receptors, IL-10Ra and IL-10Rb, it will suppress or induce the downstream cellular immune responses to protect from diseases.Materials and Methods:In this study, a potential peptide derived from IL-10 based on molecular docking and structural analysis was designed and validated by a series of cell assays to block IL-10 binding to receptor IL-10Ra for the inhibition of cell growth.Results:The simulation results indicate that the designed peptide IL10NM25 bound to receptor IL-10Ra is dominated by electrostatic interactions, whereas van der Waals (VDW) and hydrophobic interactions are minor. The cell experiments showed that IL10NM25 specifically binds to receptor IL-10Ra on the cell surface of two B-lineage cell lines, B lymphoma derived (BJAB), and lymphoblastoid cell line, whereas the mutant and scramble peptides are not able to suppress the binding of IL-10 to receptor IL-10Ra, consistent with the molecular simulation predictions.Conclusion:This study demonstrates that structure-based peptide design can be effective in the development of peptide drug discovery.

Acquisition of the Recurrent Gly101Val Mutation in BCL2 Confers Resistance to Venetoclax in Patients with Progressive Chronic Lymphocytic Leukemia

Acquisition of the Recurrent Gly101Val Mutation in BCL2 Confers Resistance to Venetoclax in Patients with Progressive Chronic Lymphocytic Leukemia

Epigenetic Modulation Leads to Re-Expression of Relapse Specific Silenced Genes and Induces Chemosensitivity in Childhood Acute Lymphoblastic Leukemia (ALL),

Abstract 3455 Introduction:The poor outcome of patients with ALL who relapse and the frequent failure of conventional salvage strategies mandate novel treatment approaches. Previous work from our laboratory led to the discovery that the histone deacetylase inhibitor (HDACi) vorinostat could effectively reverse the expression of a relapse-specific gene signature and restore blast chemosensitivity in vitro (Bhatla et al ASH 2010 #3630, ASPHO 2011 # 302D). We now have also performed genome wide DNA methylation profiling in 33 matched diagnosis/relapsed paired marrow samples by Infinium Human Methylation27 Beadchip array obtained from patients enrolled on Children’s Oncology Group (COG) protocols and observed that blasts at relapse are significantly more methylated compared those at diagnosis (p<0.00001) with concordant downregulation of gene expression. Based on these data, we sought to determine whether the demethylating agent 5-aza-2’-deoxycytidine decitabine could restore the expression of silenced genes and improve chemosensitivity of leukemic blasts and whether combination epigenetic therapy with decitabine and vorinostat could enhance further cell kill in vitro. Methods: B-lineage leukemia cell lines (Reh and UOCB1) and Ficoll enriched bone marrow patient samples (n=5) were treated with 1 μM of decitabine for 48–72 hours and quantitative RT-PCR (qRT-PCR) was performed on a panel of relapse specific, differentially methylated genes. To determine whether re-expression of genes was linked to reversal of promoter hypermethylation and not a secondary effect, genomic DNA was extracted from cell lines before and after treatment and methylation specific PCR (MSP) was performed. Unmethylated to Methylated amplicon ratios (U: M) were calculated using ImageJ software. To examine the effect of decitabine on chemosensitivity, cells were seeded in 96-well plate and treated with varying concentrations of decitabine in combination with vorinostat, prednisone, etoposide and doxorubicin. Cytotoxicity assays were performed using the Celltitre Glo Luminescent Cell Viability assay (Promega). Results were analyzed using the Calcusyn software (Biosoft) for combination drug effect which calculates a combination index (CI) where CI>1.1=antagonism, 0.9–1.1=additive and <0.90=synergy. Results: Following treatment with decitabine, we observed a 2–200 fold increase in expression by qRT-PCR, of the genes which were differentially hypermethylated and downregulated at relapse (CDKN2A, PTPRO, COL6A2, GATA4, WT1, APC and HOXA9) in primary samples and the Reh and UOCB1 leukemia cell lines. Further, MSP confirmed the enrichment of U: M amplicon ratio by 1.5 to 3 fold in these genes after decitabine treatment in cell lines. Cytotoxicity assays revealed additive or synergistic effects when primary samples and B-lineage cell lines were treated sequentially with decitabine followed by conventional chemotherapeutic agents (CI=0.5–1.05). Moreover, we observed that pretreatment with vorinostat and decitabine followed by chemotherapeutic agents had the most robust cell kill compared to any other combination (CI=0.2–0.5). Conclusions: Decitabine leads to re-expression of genes shown to be preferentially methylated at relapse and is synergistic when applied prior to conventional chemotherapy. Combination therapy with vorinostat results in maximal cell kill in cell lines and primary patient samples. Targeted epigenetic therapy with agents such as decitabine and vorinostat may be a promising approach to the treatment of relapsed ALL. Disclosures:No relevant conflicts of interest to declare.

Preferential Induction of B Cell Apoptosis Using Photodynamic Therapy.

Apoptotic cell death in cancer cells promotes antigen presentation and favors the development of an anti-tumor vaccination effect. While lymphoma cells can easily be obtained from lymph nodes or peripheral blood, these malignant cells usually co-localize with normal hematopoietic T lymphocytes, rendering the selective induction of apoptosis in malignant B cells particularly difficult. We have found that photodynamic therapy (PDT) using the photosensitizer 4–5 dibromorhodamine methyl ester (TH9402) and visible light (514 nm) can be used to preferentially eliminate malignant B-lineage non-Hodgkin's lymphoma cells (mean of 3–4 logs) over T lymphocytes. As a rhodamine derivative, TH9402 cellular efflux is mediated through the P-glycoprotein (Pgp) pump. However, the preferential elimination of B cells over resting T cells could not be ascribed to Pgp inactivation and we found no difference in intracellular retention of TH9402 between B and T cells. In order to understand the molecular events leading to such high B cell susceptibility to PDT, we then investigated the nature of death pathways involved. To determine whether apoptosis was the preponderant mechanism leading to the rapid elimination of B cells, we measured the binding of Annexin V (AnnV) in 7-aminoactinomycin D (7AAD) negative (i.e. live) B and T cells using flow cytometry, and quantified DNA fragmentation and caspase 3 and 9 activity in isolated CD3+ and CD19+ PDT treated and untreated cells. We found that B cells demonstrated high levels of apoptosis after PDT, with 53±1.3% (mean±SE) AnnexinV+/7AAD- cells compared to 30±1.9% in T cells (p< 0.05). The extent of DNA fragmentation, as determined by the TUNEL reaction, was extensive in B cells (33.7±9.8%)(mean±SE) but not T cells (2.3±0.1%) when measured at 4 hours post PDT (p<0.01). After PDT, catalytic caspase 3 levels were also 4-fold higher in CD19+ cells than CD3+ cells (p<0.01). In contrast, caspase 9 levels remained low for both cell types. The induction of apoptosis translated into the elimination of more than 90% of B cells within 4 hours after PDT, while the majority of T cells were not affected. Importantly, similar elimination of B-lineage cell lines and patient cells was observed, using a limiting dilution assay, whether cells overexpressed the anti-apoptotic bcl-2 protein or not. Thus, these results indicate that the predominant caspase 3 apoptotic pathway is specifically implicated in PDT-induced B cell death, and its early activation explains the rapid and profound sensitivity of B lymphocytes to PDT. This strategy could promote tumor cell vaccination in the context of autologous TH9402-purged stem cell transplantation for patients with B-lineage malignancies. In addition, preferential B cell targeting could take advantage of the crucial immunomodulatory role of apoptosis for the treatment of patients with autoimmune B cell disorders.

Mechanisms for T-cell selective cytotoxicity of arabinosylguanine

AbstractNelarabine, prodrug of arabinosylguanine (ara-G), has demonstrated T-lymphoblastic antileukemic activity in cell lines and in the clinic. To investigate the mechanism for lineage-specific toxicity, the effects of ara-G were compared in CEM (T-lymphoblast), Raji (B-lymphoblast), and ML-1 (myeloid) cell lines. CEM cells were the most sensitive to ara-G–induced apoptosis and accumulated the highest levels of ara-G triphosphate (ara-GTP). However, compared with myeloid and B-lineage cell lines, CEM cells incorporated fewer ara-G molecules—which were at internucleotide positions in all 3 cell lines— into DNA. Ara-G induced an S-phase arrest in both Raji and ML-1, while in CEM the S-phase cells decreased with a concomitant increase in the sub-G1 population. Within 3 hours of ara-G treatment, the levels of soluble Fas ligand (sFasL) in the medium increased significantly in CEM cultures. In parallel, an induction of FasL gene expression was observed by real-time reverse transcriptase–polymerase chain reaction (RT-PCR). Pretreatment of CEM cells with a Fas antagonistic antibody inhibited ara-G–mediated cell death. These results demonstrate that high ara-GTP accumulation in T cells results in an S phase–dependent apoptosis induced by ara-G incorporation into DNA, which may lead to a T cell–specific signal for the induction and liberation of sFasL. Subsequently, the sFasL induces an apoptotic response in neighboring non–S-phase cells. In contrast, myeloid and B cells accumulated lower levels of ara-GTP and arrested in S phase, blocking any apoptotic signaling.

FcεR1 and CD22 mRNA are expressed in early B-lineage and myeloid leukemia cell lines

CD22, one of the important markers for diagnosing B-lineage acute leukemia, was expressed in mature basophil granulocytes. We then investigated the expression of CD22 and other B cell- and basophil-related molecules in 25 human acute leukemia cell lines to find the phenotype of the virtual common progenitor of B and myeloid lineage. Surface and cytoplasmic expressions of antigens were analyzed using a flow cytometer and an essential antibody panel used for diagnosing acute leukemia as well as cytokine receptors and basophil-related enzymes. Messenger RNA expression of FcεR1 and CD22 was also analyzed. Peroxidase-positive and -negative myeloid leukemias showed eosinophil- and basophil-type expression of enzymes, respectively. Early myeloid and B-lineage cells expressed basically similar combinations of cytokine receptors and various combinations of mRNA listed above, while T-lineage cells did not. The virtual common progenitor of B and myeloid lineage cells may be defined as immature cells simultaneously expressing B and basophil phenotypes.

Supernatants of stored polymorphonuclear neutrophils exhibit growth-promoting activity in several cell lines: perforin expression in polymorphonuclear neutrophils and its role in down-regulation of growth-promoting activity.

Polymorphonuclear neutrophils (PMNs) play important roles in the host immune defence. This study was performed to identify roles of PMNs other than those already known. PMNs were separated from the peripheral blood of healthy individuals and stored in vitro for 24 h in the presence or absence of an anti-human Fc receptor (FcR) gamma III antibody, namely, anti-CD16 monoclonal antibody (mAb). Stored supernatants were harvested and incubated with several leukaemia and transformed cell lines for 48 h. The increase in growth rate was assessed by the increase in the amount of 3H-thymidine incorporated into these cells. Expression of perforin on PMNs, which is thought to decrease cell viability, was elucidated by flow cytometry (FCM) analysis. The presence of perforin in the stored supernatants was determined using an enzyme-linked immunosorbent assay (ELISA). A serine protease inhibitor, which is known to block the effect of perforin, was added to the cultures of several leukaemia and transformed cell lines to confirm the effect of perforin in reducing cell viability. Growth promotion of some cell lines cultured with the stored supernatants of PMNs was observed both in the presence and absence of anti-CD16 mAb, which was used as a trigger molecule of PMNs. This was particularly notable in the case of Raji and Daudi (both Burkitt lymphoma) cell lines and Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (B-LCLs) derived from healthy individuals. Perforin expression was observed in both freshly prepared and stored PMNs, regardless of the presence or absence of anti-CD16 mAb. ELISA also detected perforin in the stored supernatants in both the presence and absence of anti-CD16 mAb. The increase in growth rate was induced in the presence of not only a serine protease inhibitor but also an anti-perforin mAb. Stored supernatants of PMNs exhibit up-regulation of cell growth in several cell lines; this up-regulation is particularly prominent in B-lineage cell lines. Furthermore, perforin appeared to be expressed on PMNs constitutively and secreted into the extracellular fluid. Results of this study strongly suggest that the growth-promoting activity in supernatants of stored PMNs is partially inhibited by perforin, which is thought to be produced by PMNs themselves.

Long-lived polyclonal B-cell lines derived from midgestation mouse embryo lymphohematopoietic progenitors reconstitute adult immunodeficient mice.

Lymphohematopoietic progenitors derived from midgestation mouse embryos were established in long-term cultures with stromal cell monolayers and interleukin 7 (IL-7), giving rise to B-lineage cell lines. The initial emergence and in vitro establishment of these early embryo cell lines were highly sensitive to IL-7-mediated signals, in comparison to cell lines similarly obtained using precursors from late fetal liver (> 13 days postcoitum) and adult bone marrow. The early embryo-derived progenitors spontaneously differentiated in vitro to CD19(+)IgM(+) immature B cells in the presence of optimal concentrations of IL-7, in contrast to those progenitors obtained from late gestation and adult mice, whose differentiation only occurred in the absence of IL-7. The newly in vitro-generated B cells of the early embryo cell lines repopulated adult immunodeficient severe combined immunodeficient mice on their adoptive transfer in vivo and generated specific humoral immune responses after immunization.

Loss of p73 gene expression in lymphoid leukemia cell lines is associated with hypermethylation

The expression of the p73 gene and the methylation status was examined in 61 acute lymphoblastic leukemia (ALL) cell lines and lymphocytes from seven healthy individuals. p73 mRNA was not expressed in 19 (31.1%) of 61 ALL cell lines, including 11 (31.4%) of 35 B-precursor ALL cell lines, 2 (16.7%) of 12 B-ALL/Burkitt lymphoma (BL) cell lines (totally 27.7% of B-lineage cell lines), 6 (42.9%) of 14 T-ALL cell lines, and expressed in all of normal lymphocytes, by reverse transcriptase-polymerase chain reaction (RT-PCR). Restriction-enzyme related PCR (REP) and methylation-specific PCR (MSP) revealed that the cell lines lacking p73 mRNA expression were hypermethylated. In contrast, normal lymphocytes and most cell lines that expressed detectable p73 mRNA were not hypermethylated with the exception of five cell lines. Furthermore, bisulfite genomic sequencing confirmed the results obtained by REP and MSP. Our results suggest that p73 inactivation may be involved in the pathogenesis of both T- and B-ALLs, and that hypermethylation is the predominant mechanism of inactivation of the p73 gene in ALL.

Expression of thrombopoietin receptor and its functional role in human B-precursor leukemia cells with 11q23 translocation or Philadelphia chromosome

Thrombopoietin (TPO) is a hematopoietic growth factor which plays a central role in normal megakaryocytopoiesis and thrombopoiesis. Although the interaction between TPO and its receptor c-Mpl encoded by the c-mpl gene is now known to be implicated in the proliferation and/or differentiation of abnormal myeloid cells and normal hematopoietic stem cells, little is known about a role of the TPO/c-Mpl system in lymphoid leukemia cells. In the present study, we first examined the expression of c-mpl/c-Mpl in 23 human lymphoid leukemic cell lines (T-lineage 4, B-lineage 19) using three distinct methods. The c-mpl mRNA was detectable in as many as 20 cell lines (T-lineage 3, B-lineage 17) by reverse transcriptase-polymerase chain reaction, but its translated product, c-Mpl, was demonstrable by Western blot only in B-lineage cell lines. Flow cytometric analysis revealed the surface c-Mpl expression in 13 of 17 B-lineage cell lines, but its higher expression (>40%) was restricted in nine B-precursor cell lines, eight of which had 11q23 translocation or Philadelphia chromosome (Ph1). We also demonstrated that two of eight cell lines with 11q23 translocation or Ph1 exhibited a significant proliferative response to TPO in the 3H-thymidine uptake and colony-forming assays. Triggering of these cell lines by TPO transiently up-regulated tyrosine phosphorylation of JAK-2 and Shc, indicating that their receptor is functional. Primary leukemia cells separated from patients with B-precursor acute lymphoblastic leukemia with Ph1 or 11q23 translocation also showed the surface c-Mpl expression and a significant responsiveness to TPO. These results suggest that the TPO/c-Mpl interaction may play a physiological role in the growth regulation of B-precursor leukemia cells particularly with specific chromosomal abnormalities.

Characteristic expression of Hck in human B-cell precursors

Objective. To identify molecules involved in signaling for early B-cell development, we investigated the expression of signal transduction-related proteins in B-cell progenitors. Normal as well as leukemic B-cell progenitors were examined by immunoblotting and immunofluorescence study. In a survey of the expression of a broad range of signal transduction molecules, the Src-family protein tyrosine kinases were found to be differentially expressed in early B-cell differentiation. Materials and Methods. Analysis of freshly prepared precursor-B acute lymphoblastic leukemia cells and B-lineage cell lines showed Hck and Lyn are major Src-family protein tyrosine kinases expressed in this type of leukemic blasts. Results. However, heterogeneity of Hck and Lyn expression was found in these cells, and precursor-B acute lymphoblastic leukemia cells subsequently were classified according to the expression pattern of Hck and Lyn as Hck/Lyn dual-negative, Hck-predominant, Hck/Lyn dual-positive, and Lyn-predominant. Further studies on normal B-lineage cells indicated that the Src-family protein tyrosine kinases are expressed sequentially in a differentiation-dependent fashion during B-cell ontogeny and that the predominant expression of Hck is a common feature in B-cell progenitors, whereas Lyn expression is more significant in mature B cells. Conclusions. Although the biologic significance remains unknown, sequential expression of Src-family protein tyrosine kinases should play a role in regulation of early B-cell differentiation.

3′ IgH Enhancer Elements Shift Synergistic Interactions During B Cell Development

IgH gene expression is tightly controlled over the course of B cell development, B cell activation, and the subsequent differentiation of these cells into Ig-secreting plasmacytes. There are several transcriptional enhancers that map within and downstream of the IgH locus, and some of these have been clearly implicated in the developmental regulation of IgH gene assembly and expression. While some of the individual enhancers from this locus have been studied extensively, the functional interactions possible among this group of enhancers have been largely unexplored. In the present study, we have measured the transcriptional activities of combinations of enhancers introduced into B-lineage cell lines at several different developmental stages. We detected a developmental progression in which the 3' enhancers are initially inactive, then become strongly active through synergistic interactions, and finally achieve a strong level of activity with little interdependency. The relative contributions of E mu (the intron enhancer) and of the 3' enhancers also change as a function of developmental stage. We discuss these results in light of parallel studies of developmental changes in transcription factor requirements.

Contact Between Human Bone Marrow Stromal Cells and B Lymphocytes Enhances Very Late Antigen-4/Vascular Cell Adhesion Molecule-1–Independent Tyrosine Phosphorylation of Focal Adhesion Kinase, Paxillin, and ERK2 in Stromal Cells

AbstractContact with bone marrow stromal cells is crucial for the normal growth and development of B-cell precursors. We have previously shown that human bone marrow stromal cell tyrosine kinase activity can be activated by direct contact with B-lymphoid cells (J Immunol 155:2359, 1995). In the present study, we show that increased tyrosine phosphorylation of focal adhesion kinase, paxillin, and extracellular-related kinase 2 (or p42 MAP kinase) accounted for the major changes occurring in stromal cell tyrosine phosphorylation after 5 to 10 minutes of contact with the RAMOS B-lymphoma cell line. Although adhesion of B-cell precursors to stromal cells is primarily mediated by very late antigen-4 (VLA-4) and vascular cell adhesion molecule-1 (VCAM-1), VLA-4–deficient and adhesion-deficient RAMOS cells were equally capable of stimulating stromal cell tyrosine phosphorylation. Similar changes in the tyrosine phosphorylation pattern of stromal cells were induced by contact with normal human B-cell precursors and several other B-lineage cell lines. After 5 to 30 minutes of contact with stromal cells, no change in protein tyrosine phosphorylation was detected in RAMOS or normal human B-cell precursors removed from stromal cells. Pretreatment of stromal cells with cytochalasin D abrogated contact-mediated enhancement of stromal cell tyrosine phosphorylation, suggesting that an intact cytoskeleton was essential. These results suggest that B-cell contact activates stromal cell signaling cascades that regulate cytoskeletal organization and transcription, independent of the interaction mediated by VLA-4 and VCAM-1.

Contact Between Human Bone Marrow Stromal Cells and B Lymphocytes Enhances Very Late Antigen-4/Vascular Cell Adhesion Molecule-1–Independent Tyrosine Phosphorylation of Focal Adhesion Kinase, Paxillin, and ERK2 in Stromal Cells

Contact with bone marrow stromal cells is crucial for the normal growth and development of B-cell precursors. We have previously shown that human bone marrow stromal cell tyrosine kinase activity can be activated by direct contact with B-lymphoid cells (J Immunol 155:2359, 1995). In the present study, we show that increased tyrosine phosphorylation of focal adhesion kinase, paxillin, and extracellular-related kinase 2 (or p42 MAP kinase) accounted for the major changes occurring in stromal cell tyrosine phosphorylation after 5 to 10 minutes of contact with the RAMOS B-lymphoma cell line. Although adhesion of B-cell precursors to stromal cells is primarily mediated by very late antigen-4 (VLA-4) and vascular cell adhesion molecule-1 (VCAM-1), VLA-4–deficient and adhesion-deficient RAMOS cells were equally capable of stimulating stromal cell tyrosine phosphorylation. Similar changes in the tyrosine phosphorylation pattern of stromal cells were induced by contact with normal human B-cell precursors and several other B-lineage cell lines. After 5 to 30 minutes of contact with stromal cells, no change in protein tyrosine phosphorylation was detected in RAMOS or normal human B-cell precursors removed from stromal cells. Pretreatment of stromal cells with cytochalasin D abrogated contact-mediated enhancement of stromal cell tyrosine phosphorylation, suggesting that an intact cytoskeleton was essential. These results suggest that B-cell contact activates stromal cell signaling cascades that regulate cytoskeletal organization and transcription, independent of the interaction mediated by VLA-4 and VCAM-1.

Induction of murine CD5 expression by v-H-ras.

The murine CD5 surface antigen is a frequent marker on B-lineage cell lines produced from bone marrow infected with retroviruses expressing v-H-ras. Since CD5+ B cells cannot be detected in adult murine bone marrow, either the viral targets of transformation are a minor contaminating population of CD5+ B-lineage cells or the v-H-ras oncogene is inducing the expression of CD5 on B-lineage cells not previously expressing this marker. We have found that v-H-ras can induce the expression of CD5 on two CD5+ pre-B-cell lines established from murine bone marrow. This induction correlates with increased steady-state levels of CD5 mRNA. These results present the possibility that CD5 expression may be modulated by specific signalling as well as early lineage commitment.

The [formula omitted] gene is actively transcribed during all stages of murine B-lymphocyte development

It is generally accepted that the expression of FcϵRII CD23 on the surface of B-lineage cells is restricted to the stage of the resting, mature ( sIgM + sIgD + ) B-lymphocyte. However, it is unknown whether activation of the FcϵRII CD23 gene is also restricted to the stage of the mature B-lymphocyte. To address this question we investigated a panel of B-lineage cell lines for the presence of transcripts encoding FcϵRII CD23 . We detected transcripts in 16 of 26 B-lineage cell lines representing the entire spectrum of B-cell development. In most cases (13 of 16) active transcription of the murine FcϵRII CD23 gene was not coupled with the expression of cell surface FcϵRII CD23 protein. The stage-restricted pattern of cell surface FcϵRII CD23 expression did not hold for all murine B-cell lines. One post-switch B-cell line ( sIgM − sIgG + ) expressed FcϵRII CD23 on the cell surface and another could be induced with IL-4 and LPS to express surface FcϵRII CD23 . Transcription of the murine CD23 gene in the absence of cell surface expression of FcϵRII CD23 does not appear to simply be an aberrant feature of transformed B-cells since we found transcripts, but not surface expression, in some normal splenic and peritoneal B-lymphocytes. Our findings suggest that the potential for expression of FcϵRII CD23 may occur over a much broader developmental window of the B-lineage than previously suspected. Transcription of the FcϵRII CD23 gene, in the absence of detectable cell surface protein expression in B-lineage cell lines, and in sort-purified B-lymphocyte subpopulations, implies that in addition to regulatory mechanisms already known, murine CD23 is also regulated through post-transcriptional mechanisms that have not yet been characterized.

Cytokine-Mediated Regulation of Transferrin Synthesis in Mouse Macrophages and Human T Lymphocytes

Transferrin (Tf) plays an important role during immunologic activation by donating iron to activated lymphocytes. Therefore, synthesis by lymphomyeloid cells has been investigated. Mouse macrophages and macrophage cell lines synthesized Tf, with levels being markedly increased by gamma-interferon (gamma-IFN) and, to a lesser extent, by interleukin-1 beta (IL-1 beta), IL-6, and tumor necrosis factor alpha (TNF alpha). Tf was also produced by phytohemagglutinin-stimulated human T cells and two T-cell lines and was increased by IL-2. Even after appropriate activation, none was synthesized by human macrophages or monocytic cell lines or by mouse T cells, T-cell lines, or thymus cells. In both species, B-lineage cell lines were negative. Tf was also synthesised by macrophages from congenitally hypotransferrinemic mice and was responsive to gamma-IFN, but levels were lower than those from normal controls. Synthesis by human and murine hepatoma cells was increased by IL-6 but unaffected by IL-1, TNF alpha, or gamma-IFN. Iron decreased synthesis by hepatoma cells but had no effect on the lymphomyeloid cells. Tf mRNA levels paralleled protein synthesis, suggesting that regulation was pre-translational. Thus, Tf synthesis by lymphomyeloid cells is regulated differently from hepatic synthesis, which is consistent with the suggestion that Tf may act in a paracrine (mouse) or autocrine (human) manner on activated lymphocytes.

Sequential induction of NF-kappa B/Rel family proteins during B-cell terminal differentiation.

The NF-kappa B/Rel family of at least five transcription factor polypeptides is thought to function both as a developmental regulator in B cells and as a rapid response system in all cells. To examine this notion in more detail, we determined the protein contents of both the inducible and constitutive NF-kappa B/Rel activities in a pre-B-cell line, 70Z/3, and a mature B-cell line, WEHI 231. NF-kappa B p50/p65 is the major inducible nuclear complex after lipopolysaccharide or phorbol myristate acetate treatment of 70Z/3 cells. The constitutive and inducible complexes in WEHI 231 cells are mainly composed of p50 and Rel. The constitutive or induced activities are all sensitive to I kappa B-alpha, but this inhibitor is very short-lived in WEHI 231 cells, suggesting that the balance between synthesis and degradation of I kappa B-alpha determines whether a particular cell lineage has constitutive activity. A patterned expression of the NF-kappa B/Rel activator proteins emerges from an analysis of other B-lineage cell lines and splenic B cells: mainly p50 and p65 in pre-B (and non-B) cells, a predominance of Rel and p50 in mature B cells, and expression of p52 and RelB in plasmacytoma lines. This ordered pattern of regulators may reflect the requirement for expression of different genes during terminal B-cell differentiation because different combinations of NF-kappa B/Rel family members preferentially activate distinct kappa B sites in reporter constructs.

Sequential induction of NF-kappa B/Rel family proteins during B-cell terminal differentiation.

The NF-kappa B/Rel family of at least five transcription factor polypeptides is thought to function both as a developmental regulator in B cells and as a rapid response system in all cells. To examine this notion in more detail, we determined the protein contents of both the inducible and constitutive NF-kappa B/Rel activities in a pre-B-cell line, 70Z/3, and a mature B-cell line, WEHI 231. NF-kappa B p50/p65 is the major inducible nuclear complex after lipopolysaccharide or phorbol myristate acetate treatment of 70Z/3 cells. The constitutive and inducible complexes in WEHI 231 cells are mainly composed of p50 and Rel. The constitutive or induced activities are all sensitive to I kappa B-alpha, but this inhibitor is very short-lived in WEHI 231 cells, suggesting that the balance between synthesis and degradation of I kappa B-alpha determines whether a particular cell lineage has constitutive activity. A patterned expression of the NF-kappa B/Rel activator proteins emerges from an analysis of other B-lineage cell lines and splenic B cells: mainly p50 and p65 in pre-B (and non-B) cells, a predominance of Rel and p50 in mature B cells, and expression of p52 and RelB in plasmacytoma lines. This ordered pattern of regulators may reflect the requirement for expression of different genes during terminal B-cell differentiation because different combinations of NF-kappa B/Rel family members preferentially activate distinct kappa B sites in reporter constructs.