Biomedical subjects

J G Monroe

Publications and source records attributed to J G Monroe.

At least 19 recordsLinked to original sources

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↗

PubMed1995

src-family tyrosine kinase p55fgr is expressed in murine splenic B cells and is activated in response to antigen receptor cross-linking.

Stimulation of the B cell Ag receptor (BCR) triggers a complex cascade of intracellular signals that lead to activation, proliferation, and differentiation. One of the earliest and most critical events following Ag receptor cross-linking is the activation of receptor-associated tyrosine kinases, which phosphorylate a variety of substrates involved in transducing signals generated through the receptor. Several tyrosine kinases have been shown to be associated with the BCR, including the src family members Lyn, Fyn, Blk, and Lck and the non-src kinase Syk. Here we describe another src family kinase expressed in B cells, p55fgr. Although previous studies have suggested that Fgr expression in the mouse is restricted to cells of the myeloid lineage, we demonstrate that highly purified murine splenic B cells express the kinase as well. We also show that Fgr, like other B cell kinases, is activated in response to cross-linking of the Ag receptor. Unlike these other kinases, however, Fgr does not appear to be physically associated with the receptor, and does not become activated until several minutes after stimulation. Moreover, we find that Fgr is associated with a distinct set of cellular proteins. On the basis of these studies, we hypothesize that Fgr may play a unique role in the BCR-associated signaling pathway.

Animals↗

PubMed1995

Transient transfection of murine B lymphocyte blasts as a method for examining gene regulation in primary B cells.

Studies of the biochemical and genetic processes associated with activation of B lymphocytes have contributed much to the understanding of the regulation of the B cell response to antigen. Primary, non-transformed B cells from the spleen in mice and the tonsils or peripheral blood in humans have proven to be informative models for dissection of the biochemical events leading to B cell activation. In contrast, genetic studies of this process have relied on transformed cell lines grown in culture. The influence of the transformed state on the results obtained using these models may limit their physiological relevance. This report describes a method whereby non-transformed B lymphocytes in primary culture can be transfected for use in studies of gene regulation in response to antigen receptor signals. Transfection was accomplished after only a 72 h exposure to LPS. The cells obtained after LPS treatment were greater than 97% pure, and more importantly, remained responsive to antigen-receptor generated signals. Responsiveness was confirmed by demonstrating induction of mRNA for the primary response gene egr-1, as well as induction of specific transcription factor binding activity in nuclear extracts from these cells. DEAE-dextran-mediated transient transfection was utilized to introduce an egr-1 promoter/reporter construct into these cells. This analysis of promoter activity yielded results which were indistinguishable from the pattern of expression of the endogenous egr-1 gene. Potential applications for dissection of transcriptional regulatory pathways in B lymphocytes are discussed.

Animals↗

PubMed1995

Immature B lymphocytes are deficient in expression of the src-family kinases p59fyn and p55fgr1.

Unlike mature B cells, immature B cells are highly sensitive to tolerance induction and do not proliferate in response to stimulation through their Ag receptors. Our previous studies have suggested that this differential responsiveness is caused by differences in Ag receptor-proximal signaling pathways. Because many of these pathways are regulated by tyrosine phosphorylation, we have compared the expression and activity of tyrosine kinases in immature and mature B cells. Consistent with previous studies, we demonstrate that mature B cells express syk, lyn, blk, hck, and fyn; in addition, we find that they express significant amounts of the src-family kinase p55fgr. Immature B cells from day-3 mouse spleen show similar expression and activity of syk, lyn, blk, and hck. However, these cells have marked deficiencies in fyn and fgr expression. Differential expression of fyn and fgr is also observed in immature cells from the bone marrow, indicating that this phenotype is characteristic of the immature stage of B cell development. High levels of fyn and fgr are detectable in splenic B cells beginning at 4 wk of age, coincident with the appearance of cells that proliferate in response to receptor cross-linking. These observations indicate that expression of the src-family kinases fyn and fgr is regulated developmentally in murine B lymphocytes. Moreover, they suggest that these kinases may be important for mature B cell responses to Ag receptor stimulation, and that their absence may contribute to the unresponsiveness and tolerance susceptibility of immature B cells.

Animals↗

PubMed1995

Activation of the p21ras pathway couples antigen receptor stimulation to induction of the primary response gene egr-1 in B lymphocytes.

The primary response gene egr-1 encodes a sequence-specific transcription factor whose expression is necessary for antigen receptor-stimulated activation of B lymphocytes. The molecular processes involved in linking egr-1 induction to antigen receptor signaling have not been defined. The present study demonstrates that expression of an activated form of p21ras results in egr-1 induction similar to that previously shown after antigen receptor cross-linking. In addition, both antigen receptor cross-linking and p21ras use the same element in the egr-1 promoter to exert their effects. Using dominant-negative mutants of p21ras and raf-1, we demonstrate that induction of egr-1 after antigen receptor cross-linking is mediated by activation of the p21ras/mitogen-activated protein kinase signaling pathway. While regulation of the p21ras pathway during B cell activation has been intensively studied, this report represents the first description of a biologically relevant event associated with its activation.

Animals↗

PubMed1995

HIV-mediated B-lymphocyte activation and lymphomagenesis.

Non-Hodgkin's (1ii)lymphoma is an AIDS-defining event in a significant percent of U.S. patients infected with the human immunodeficiency virus (HIV). Advances in anti-retroviral treatment and management of opportunistic infection have been accompanied by an increase in the incidence of these lymphomas. In the immunocompromised state of patients late in the course of HIV infection, these lymphomas represent a complication of HIV infection that is associated with an extremely poor prognosis. Currently, there is little understanding of the pathogenesis of HIV-associated lymphomas, which are nearly exclusively of B-cell origin. Experimental data do not support HIV infection in these lymphomas. While some lymphomas show evidence of EBV infection, the majority do not. Polyclonal B-cell hyperactivity during the early phases of HIV infection argues that chronic B-cell stimulation may be the major process predisposing B-cells in the HIV-infected individual to malignant transformation. The mechanism of this stimulation of normal B cells and its relationship to AIDS-associated lymphomas remain poorly understood. In this review, we will summarize current information on HIV-associated B lymphoma and then discuss our views on the association and regulation of HIV-related hyperactivity on the pathogenesis of this lymphoma.

B-Lymphocytes↗

PubMed1995

The EGR1 protein contains a discrete transcriptional regulatory domain whose deletion results in a truncated protein that blocks EGR1-induced transcription.

Egr-1 is a ubiquitous immediate-early gene whose expression is induced by a wide range of different stimuli. A requirement for egr-1 expression has been demonstrated in pathways leading to both proliferation and differentiation, suggesting that egr-1 is a critical intermediary in determining the long-term cellular response to a stimulus. To determine how egr-1 coordinates a cellular response to receptor-mediated stimulation, we have developed a transient cotransfection assay to map functional domains in the EGR1 protein. We localized an activation domain to a serine/threonine/proline-rich region between amino acids 174 and 270. Using this information, we designed a mutant that lacks this activation domain, but retains the DNA-binding domain. When cotransfected into fibroblasts with an EGR1-dependent reporter, this mutant inhibited the transcriptional activity of both endogenous EGR1, as well as exogenously expressed, wild-type EGR1 protein. These data demonstrate that the activation domain of EGR1 is critical for the activity of the protein, and that a mutant lacking this domain can dominantly inhibit wild-type EGR1 function.

3T3 Cells↗