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Biomedical subjects

J G Monroe

Publications and source records attributed to J G Monroe.

At least 19 recordsLinked to original sources
PubMed1998

Antigen receptor-initiated signals for B cell development and selection.

The B cell antigen receptor or signaling components of this receptor play varying roles in determining the fate of the B cell at different stages of B cell development. Signals generated through this receptor complex or its various components determine the survival, progression, expansion, and activation of the B cell. Factors that determine the fate of the B cell following antigen receptor engagement include the developmental stage, coreceptor expression, degree and stability of ligand-receptor engagement, and the availability of T cell-derived secondary signals. The author's laboratory is interested in defining the molecular processes linking B cell antigen receptor signaling to these specific cellular responses in the context of antigen-independent and dependent B cell development. Here our current progress and thinking with regard to the determination between negative selection and activation as it pertains to the transition from the immature- to the mature-stage B cell will be discussed.

Animals↗
PubMed1998

B cell receptor-induced apoptosis in primary transitional murine B cells: signaling requirements and modulation by T cell help.

Self-reactive immature B cells may be eliminated in the bone marrow (BM) after B cell receptor (BCR) engagement in a process known as negative selection. Immature B cells emigrating from the BM, the so-called transitional cells, remain sensitive to negative selection and are likely to be important targets of tolerance towards peripheral antigens. Transitional cells are deleted through apoptosis after BCR cross-linking in vitro. Using anti-Ig as a surrogate antigen, we determined the signaling requirements for the induction of apoptosis in transitional cells. Treatment with anti-Ig for only 20 min causes most cells to be apoptotic 16 h later. Furthermore, apoptosis of transitional cells is induced with low doses of anti-Ig while mature cell proliferation requires extended culture at 30-fold higher concentrations. For both populations of B cells, total surface Ig expression is equivalent, therefore indicating that the threshold of BCR signaling required to elicit these responses is different. T cell help can modulate B cell tolerance. However, specific help may not be available when apoptosis is triggered by a peripheral antigen. The opportunity to reverse apoptosis of transitional cells is surprisingly long. Even 8 h after anti-Ig treatment, IL-4 or anti-CD40 antibody can block apoptosis. The upper time limit of protection is concurrent with irreversibility of apoptosis as measured by DNA fragmentation. These findings indicate that B cell negative selection is more easily triggered than activation, and that the induction of apoptosis and its reversal by T cell help can be events that occur in distinct microenvironments.

Animals↗
PubMed1997

Characterization of anti-single-stranded DNA B cells in a non-autoimmune background.

Anti-DNA Abs are prevalent in the serum of systemic lupus erythematosus (SLE) patients and in the MRL-lpr/lpr mouse model of SLE, but are generally absent in normal individuals. We have studied the regulation of anti-ssDNA B cells in a non-autoimmune (BALB/c) background by using Ig transgenes (Tgs) encoding anti-DNA Abs. In one case, they are present with other non-DNA-binding B cells (the VH3H9 Tg with endogenous light chains); in the other, they are present as an essentially monospecific population (VH3H9/Vkappa8). We have previously observed that serum anti-ssDNA levels in these Tg mice were no higher than those of non-Tg mice, despite the fact that anti-ssDNA B cells dominate the peripheral B cell repertoire. These results suggested that the anti-ssDNA Tg B cells present are functionally inactivated. In this paper, we isolate B cells from VH3H9/Vkappa8 Tg mice to show that this is indeed the case and go on to further define this state. We demonstrate that VH3H9/Vkappa8 Tg B cells have diminished Ig secretion in response to both T-independent and T-dependent stimuli compared with non-Tg controls. VH3H9/Vkappa8 Tg B cells also show suboptimal proliferation in response to anti-IgM F(ab)'2 fragments and LPS, and are phenotypically distinct in expressing decreased total surface Ig. Despite their functional defects, however, VH3H9/Vkappa8 Tg B cells have an in vivo turnover rate comparable to non-Tg B cells, suggesting that they are long lived.

Animals↗
PubMed1997

Biochemistry of antigen receptor signaling in mature and developing B lymphocytes.

The function of the surface antigen receptor of B cells (BCR) has been extensively studied with respect to the activation of mature B lymphocytes. B cells at other points of development (e.g., pre-B cells, immature B cells, and germinal center B cells) also express forms of the BCR, and for cells at these developmental timepoints, signaling through the BCR in some cases may lead to outcomes other than B-cell activation. Understanding the molecular events that are initiated by BCR crosslinking would enhance our understanding of the regulation and functional results of BCR signaling throughout B-cell development. In this article we review the current understanding of BCR signal transduction from initiation of the signal through changes in expression of genes that regulate the activation state. Costimulatory and modulatory molecules are considered with regard to their ability to affect the sensitivity or outcome of the BCR signal. Finally, we discuss how BCR signal transduction and the results of BCR signaling may differ at distinct stages in B-cell development.

Animals↗
PubMed1997

Decreased immunoglobulin deposition in tumors and increased immature B cells in p53-null mice.

Recent studies have hinted that there may be a relationship between p53 and the immune response. In preliminary experiments, we found significantly decreased levels of immunoglobulin deposition in 13 of 16 p53-null tumors compared with 2 of 17 tumors derived from p53 +/- mice. We further explored the effect of p53 on B-cell development and function. p53-null mice contained more splenic white pulp and more immature B cells in the bone marrow compared with p53 +/- mice. p53-null B cells were hyperresponsive to proliferative challenge but were not more resistant to signal-induced apoptosis. Several p53 DNA-binding sites were localized to the regulatory regions of immunoglobulin heavy and light chain genes, including the KII site, which serves as an enhancer for rearrangement of the mouse kappa chain J cluster genes. Levels of p53 protein and the kappa chain sterile transcript increased after exposure of pre-B cells to the DNA damaging agents etoposide and Adriamycin. Our observations suggest that p53 may be involved in B-cell maturation and may relay certain stress signals to affect B-cell function.

Animals↗
PubMed1996

Evidence for early B-cell activation preceding the development of Epstein-Barr virus-negative acquired immunodeficiency syndrome-related lymphoma.

To investigate the origin and pathogenesis of acquired immunodeficiency syndrome (AIDS)-related lymphoma (ARL), we studied 14 cases in which Epstein-Barr virus (EBV) infection was not an etiologic factor. By histology, 8 of the specimens were of the small noncleaved cell type and 6 consisted of the large diffuse cell type. Southern analysis using a J(H) probe was consistent with a monoclonal B-cell tumor in 13 cases. To characterize the expressed Ig genes, we performed reverse transcriptase-polymerase chain reaction (RT-PCR) and direct sequencing of PCR products. Eight cases expressed IgM and 1 case expressed IgG. V(H)3 genes were found in 5 cases, V(H)4 genes in 3 cases, V(H)1 genes in 2 cases, and a V(H)7 gene in 1 case. The nucleotide homology to known germline V(H) genes ranged from 80% to 97%, suggesting significant somatic diversification of expressed V(H) genes. The large proportion of V(H)3-expressing lymphomas in this series corresponds to the frequency of V(H)3-expressing B cells in the peripheral blood from healthy and (recent) human immunodeficiency virus (HIV)-seropositve individuals and contrasts with the V(H)3 clonal deficit observed in late stages of HIV infection. Similar to the Ig heavy chain genes, the corresponding Ig light chain genes showed significant deviation from known germline gene sequences. The large proportion of V(H)3-expressing lymphomas as well as the high degree of somatic deviation from germline suggest that these EBV-negative lymphomas might arise from antigen-selected expanded B-cell clones before transformation. Further support for this hypothesis is provided by sequential Ig sequence analysis in 1 patient with large-cell lymphoma. It was shown that 3 years before the diagnosis of axillary lymphoma, there existed several B-cell clones in this patient's bone marrow. One of these clones present in the bone marrow expressed the same rearranged V(H) gene as the axillary lymphoma. Taken together, the current findings from Ig gene analyses suggest that activation of B cells in the early phase of HIV infection may be a predisposing factor for subsequent B-cell transformation.

Amino Acid Sequence↗
PubMed1996

Activation of mitogen-activated protein kinase couples neurotensin receptor stimulation to induction of the primary response gene Krox-24.

Neurotensin (NT) is a neuropeptide that is important in a variety of biological processes such as signal transduction and cell growth. NT effects are mediated by a single class of cell-surface receptors, known as neurotensin receptors (NTRs), which exhibit structural features of the G-protein-coupled receptors superfamily. We investigated NTR signalling properties with Chinese hamster ovary (CHO) cells stably transformed with human NTR (hNTR). First, we showed that NTR stimulation by NT induced the activation of the mitogen-activated protein kinases (MAPKs) in time- and dose-dependent manners. Both p42 and p44 MAPK isoforms were retarded in gel-shift assays, which was consistent with their activation by phosphorylation. In addition we showed that NT caused a prolonged activation of MAPK as measured by in-gel kinase assay. Secondly, we demonstrated that NT induced the expression of the growth-related gene Krox-24 at the protein level, as assessed by Western-blot analysis, and at the transcriptional level, as demonstrated in CHO cells transfected with hNTR and a reporter gene for Krox-24. Activation of MAPK and induction of Krox-24 were both prevented by the NTR antagonist SR 48692, confirming the specific action on NTR. Furthermore we observed coupling of NTR to a mitogenic pathway and Krox-24 induction in the human adenocarcinoma cell line HT29, which naturally expresses NTRs. Considering coupling pathways between NTR stimulation and MAPK activation, we observed a partial inhibition by pertussis toxin (PTX) and a complete blockade by the protein kinase C (PKC) inhibitor GF 109203X. Taken together, these results suggest that (1) stimulation of NTR activates the MAPK pathway by mechanisms involving dual coupling to both PTX-sensitive and PTX-insensitive G-proteins as well as PKC activation, and (2) these effects are associated with the induction of Krox-24, which might be a target of MAPK effector.

Animals↗
PubMed1996

Immature stage B cells enter but do not progress beyond the early G1 phase of the cell cycle in response to antigen receptor signaling.

In contrast to mature B cells, immature stage B cells do not proliferate following Ag receptor cross-linking with anti-Ig Abs. To determine where in the cell cycle immature B cells arrest, we have examined the expression of specific G, cell cycle regulators. Following surface IgM (sIgM) cross-linking on mature B cells, we observed increased expression of the early G1 kinase, cyclin-dependent kinase 4 (cdk4), and one of its regulatory subunits, cyclin D2. Mature B cells also showed increased expression of components required for G1/S transition, including cyclin E and cdk2. Whereas immature stage B cells increased expression of cyclin D2 and cdk4 after anti-IgM stimulation, unlike mature stage B cells they failed to express cyclin E and cdk2. Expression of cyclin D2 and cdk4 indicates that these cells can exit G0 and enter the initial G1 phase following sIgM ligation. Interestingly, IL-4, which by itself does not stimulate proliferation of immature B cells, induced expression of cyclin E and cdk2. These latter results suggest that IL-4 complements sIgM, signaling for proliferation by increasing the basal levels of late G1 cell cycle regulators. Consistent with this idea, IL-4 synergizes with anti-Ig Abs to promote cell cycle progression and proliferation of immature B cells. Finally, c-myc, a transcriptional regulator of some members of the cell cycle machinery, is not induced following sIgM cross-linking of immature cells. This lack of inducible expression contrasts with that seen in mature stage B cells, and in immature stage cells stimulated to proliferate with LPS. These results suggest that c-myc may be a component of the signaling pathway that induces cyclin E and cdk2 expression.

Animals↗
PubMed1996

Tolerance sensitivity of immature-stage B cells: can developmentally regulated B cell antigen receptor (BCR) signal transduction play a role?

During development, B lymphocytes pass through a window in which self-reactive cells are eliminated. Elimination at this early stage of development occurs because immature-stage B cells are more sensitive to tolerance induction and negative selection than are mature-stage B cells. In this discussion, the author presents the arguments leading to the conclusion that the molecular basis for this differential sensitivity to negative selection is due to intrinsic differences in Ag receptor signal transduction between immature and mature B cells.

Animals↗
PubMed1996

Transcriptional regulation of the Icam-1 gene in antigen receptor- and phorbol ester-stimulated B lymphocytes: role for transcription factor EGR1.

Intercellular adhesion molecule (ICAM) 1/CD54 plays an important role in T cell dependent B cell activation and for function of B lymphocytes as antigen-presenting cells. ICAM-1 expression is upregulated as a consequence of B lymphocyte antigen receptor (BCR) signaling, thereby serving to render antigen-stimulated B cells more receptive to T cell-mediated costimulatory signals. We have investigated BCR-induced expression of the Icam-1 gene in primary B cells and B cell lines and have found it to be dependent on BCR-induced expression of the transcription factor EGR1. Icam-1 transcription, induced by BCR cross-linking or bypassing the BCR with phorbol ester, is absent in a B cell line in which the EGR1-encoding gene (egr-1) is methylated and not expressed. A potential EGR1-binding site was located at -701 bp upstream of the murine Icam-1 gene transcription start site and shown by electrophoretic mobility shift assay to bind to murine EGR1. Mutation of this site in the context of 1.1 kb of the Icam-1 promoter significantly abrogated transcriptional induction by phorbol ester and anti-mu stimulation in primary B cells. A direct effect of EGR1 on the Icam-1 promoter is suggested by the ability of EGR1 expressed from an SV40-driven expression vector transactivate the wild-type Icam-1 promoter, whereas mutation of the EGR1 mutation of the EGR1 binding motif at -701 bp markedly compromises this induction. These data identify EGR1 as a signaling intermediate in BCR-stimulated B cell functional responses, specifically linking BCR signal transduction to induction of the Icam-1 gene. Furthermore, similar findings for BCR-induced CD44 gene induction (Maltzman, J.S., J.A. Carman, and J.G. Monroe. 1996. Role of EGR1 in regulation of stimulus-dependent CD44 transcription in B lymphocytes. Mol. Cell. Biol. In press) suggest that EGR1 may be an important signaling molecule for regulating levels of migration and adhesion molecules during humoral immune responses.

Animals↗
PubMed1996

Acquisition of surface IgD fails to protect from tolerance-induction. Both surface IgM- and surface IgD-mediated signals induce apoptosis of immature murine B lymphocytes.

While mature splenic B cells are surface (s)IgM+ and sIgD+, immature, tolerance-susceptible B cells express sIgM and varying levels of sIgD. Differential expression of sIgD on tolerance-susceptible and resistant B cells suggests that sIgM and sIgD may transmit qualitatively different signals. Alternatively, tolerance sensitivity may be associated with intrinsic differences in sIg signaling, regardless of the isotype engaged. Here, we have exploited a stage of B cell development at which immature, tolerance-sensitive B cells co-express sIgD and sIgM. Using these immature stage B cells to evaluate isotypic differences in the ability to induce activation and deletion, we have found that neither ligation of sIgD nor sIgM is capable of inducing proliferation. Moreover, in contrast to mature B cells, both sIgD and sIgM induce apoptosis by immature stage B cells. Importantly, cross-linking sIgD does not protect immature B cells from sIgM-induced apoptosis. Thus, the differences in tolerance susceptibility of immature and mature B cells must be due to intrinsic developmental rather than isotypic differences in Ag receptor signal transduction.

Animals↗
PubMed1996

The role of early growth response gene 1 (egr-1) in regulation of the immune response.

The induction of immediate early genes in cells of the immune system is critical to determining the ultimate outcome of exposure to antigen. The importance of many of these genes relates to the role their transcription factor products play in dictating patterns of expression of downstream, function-related genes. Evidence from several systems indicates that the immediate early gene, egr-1 may be of particular importance in the immune system. Recently, the egr-1 promoter has been shown to be highly responsive to the diverse biochemical signals generated by antigen and cytokines in cells of the immune system. Furthermore, an important role for egr-1 in determining the differentiation pathway of myeloid cell precursors has been recently elaborated. Finally, potential targets of regulation by the zinc-finger transcription factor encoded by egr-1 include the interleukin-2, CD44, ICAM-1, and tumor necrosis factor genes. The role of egr-1 in regulation of the immune response will be discussed in the context of these recent studies.

Animals↗
PubMed1996

Use of isolated immature-stage B cells to understand negative selection and tolerance induction at the molecular level.

Encounter with antigen by newly developing antigen receptor-positive B cells leads to negative selection. This process positions the B cell antigen receptor (BCR) in a central role for initiating the process of negative selection and suggests developmental regulation of its signaling. The observation that immature B cells are more susceptible to negative selection than are mature B cells has been demonstrated in a number of in vitro and in vivo model systems and support the idea of developmental regulation of BCR-initiated responses. Since identical antigen receptors are expressed on immature and mature B cells, the critical fate-determining distinction between these developmental stages must lie downstream of the receptor-ligand interaction itself, in the form of different BCR-linked signaling processes or with different secondary events occurring subsequent to BCR cross-linking. To address the first possibility, our laboratory and others have sought to define the differences in BCR-mediated signal transduction in immature and mature B lymphocytes. In this review article we will discuss current in vitro systems to study this question in primary, nontransformed murine B lymphocytes. In addition, we will discuss our previously published work in order to illustrate how these model systems have been useful in beginning to unravel the molecular basis for immune B cell negative selection and tolerance.

Animals↗
PubMed1996

Lipopolysaccharide prevents apoptosis and induces responsiveness to antigen receptor cross-linking in immature B cells.

Unlike mature B cells, immature B cells are not activated in response to antigen receptor cross-linking. To examine the mechanisms underlying this unresponsiveness, we have studied the effects of reagents that have been shown to alter the responses of immature B cells to antigen receptor stimulation. Bacterial lipopolysaccharide (LPS) is a polyclonal B-cell activator, and has been shown to interfere with B-cell tolerance induction in vivo and in vitro. Here we show that LPS can also overcome the unresponsiveness of immature B cells to stimulation with anti-receptor (anti-mu) antibodies. LPS synergizes with anti-mu to induce a proliferative response that exceeds the response of immature B cells to LPS alone. Moreover, pretreatment of immature cells with LPS allows them to proliferate in response to subsequent stimulation with anti-mu antibodies. This induction of responsiveness to anti-mu requires exposure to LPS for at least 8 hr. Although the mechanisms of induction are not fully understood, one component of the LPS effect appears to involve enhancement of immature B-cell survival in culture. Neonatal splenic B cells undergo spontaneous apoptosis at a much higher rate than mature B cells, but we have found that LPS causes a dramatic inhibition of apoptosis, even when it is present for only the first 8 hr of culture. The ability of LPS to promote survival of immature B cells and allow them to proliferate in response to antigen receptor stimulation provides a system for investigation of the biochemical mechanisms of unresponsiveness and tolerance susceptibility.

Animals↗
PubMed1996

Role of EGR1 in regulation of stimulus-dependent CD44 transcription in B lymphocytes.

The immediate-early gene egr-1 encodes a transcription factor (EGR1) that links B-cell antigen receptor (BCR) signals to downstream activation events through the regulation of previously unidentified target genes. Here we identify the gene encoding the lymphocyte homing and migration protein CD44 as a target of EGR1 regulation in B cells. BCR-induced increases in CD44 mRNA expression and transcription levels are shown to occur in EGR1-expressing but not in nonexpressing subclones of the B-cell line WEHI-231. Kinetics of egr-1 transcription and the appearance of nuclear EGR1 protein precede CD44 induction and occur within 30 min after stimulation in the EGR1-expressing subclone. A single EGR1 binding motif is demonstrated at bp -301 of the human CD44 promoter. Cotransfection of a CD44 promoter-chloramphenicol acetyltransferase reporter construct with an egr-1 expression vector resulted in a 6.5- to 8.5-fold induction of transcriptional activity relative to an empty expression vector. The EGR1 binding motif was shown to be necessary for stimulus-induced expression of a CD44 promoter-chloramphenicol acetyltransferase reporter construct in nontransformed B lymphocytes and was required for transactivation by an EGR1 expression vector in a B-cell line. These studies identify EGR1 as an intermediary linking BCR-derived signals to the induction of CD44. The relevance of these molecular events to BCR signal transduction and antigen-stimulated B-cell-mediated immune responses is discussed.

Animals↗
PubMed1995

Association of p72syk with the src homology-2 (SH2) domains of PLC gamma 1 in B lymphocytes.

Phospholipase C gamma-catalyzed inositol phospholipid hydrolysis, a critical step in B cell antigen receptor signaling leading to second messenger generation and proliferation, depends upon tyrosine kinase activation. The B cell antigen receptor-associated tyrosine kinases p53/56lyn, p59fyn, p55blk, and p72syk are assumed to participate in receptor-initiated signaling. It is unknown, however, which of these kinases is involved in the tyrosine phosphorylation and resulting activation of phospholipase C gamma in response to antigen receptor cross-linking. We have used a fusion protein containing the tandem src homology-2 (SH2) domains of phospholipase C gamma 1 (PLC gamma 1) to identify B cell kinases which associate with PLC gamma 1. Using an in vitro kinase assay, we demonstrate SH2-dependent association of tyrosine kinase activity from anti-mu-stimulated B cells. The PLC gamma 1 SH2 domains associate with a prominent 70-72-kDa tyrosine phosphoprotein from anti-mu-stimulated, but not resting, B cells. Immunoblotting and secondary immunoprecipitation studies definitively identify this protein as p72syk. These results imply a physical interaction between PLC gamma 1 and p72syk in antigen receptor-stimulated B cells. This conclusion is confirmed by our ability to co-immunoprecipitate p72syk and PLC gamma 1 from lysates of anti-mu-stimulated B cells. These results implicate p72syk in the activation of phospholipase C gamma 1 during B cell antigen receptor signaling.

Animals↗
PubMed1995

Engagement of the antigen-receptor on immature murine B lymphocytes results in death by apoptosis.

During their development B lymphocytes pass through a maturational stage in which encounter with Ag leads to tolerance rather than activation. At least four mechanisms for achieving B cell tolerance have been reported: deletion, anergy, receptor editing, and competition for follicular niches. Although turnover rates for immature B cells in the adult mouse bone marrow and several transgenic model systems suggest that a major process contributing to negative selection of B cells is deletion, a detailed study of the negative effect of Ag-receptor engagement on primary, immature B cell survival has never been undertaken. We have utilized an in vitro culture system to determine whether cross-linking sIgM on tolerance-susceptible sIgM+IgD- B cells results in deletion by apoptosis. In contrast to the effect of sIgM cross-linking on mature splenic B cells, treatment of immature, bone marrow-derived B cells results in significant levels of apoptotic death. Ag receptor-mediated apoptosis is detectable by 14 h after sIgM engagement. Moreover, IL-4 and cycloheximide, which have previously been shown to prevent B cell tolerance induction, specifically block the sIgM-induced apoptosis observed in the immature B cells. Similarly, immature B cells from the neonatal spleen are also susceptible to apoptosis after sIgM cross-linking, although they manifest somewhat higher levels of unstimulated apoptosis as compared with bone marrow-derived B cells. These studies are the first detailed demonstration of Ag receptor-mediated apoptosis of primary immature stage B lymphocytes.

Animals↗