John G. Monroe

ITAM-mediated tonic signalling through pre-BCR and BCR complexes

Studies carried out over the past few years provide strong support for the idea that Igα–Igβ-containing complexes such as the pre-B-cell receptor and the B-cell receptor can signal independently of ligand engagement, and this has been termed tonic signalling. In this Review, I discuss recent literature that is relevant to the potential mechanisms by which tonic signals are initiated and regulated, and discuss views on how tonic and ligand-dependent (aggregation-mediated) signalling differ. These mechanisms are relevant to the possibility that tonic signals generated through immunoreceptor tyrosine-based activation motif (ITAM)-containing proteins that are expressed by oncogenic viruses induce transformation in non-haematopoietic cells.

Negative Selection of Immature B Cells by Receptor Editing or Deletion Is Determined by Site of Antigen Encounter

Immature B cells that encounter self-antigen are eliminated from the immune repertoire by negative selection. Negative selection has been proposed to take place by two distinct mechanisms: deletion by apoptosis or alteration of the antigen receptor specificity by receptor editing. While convincing evidence exists for each, the two models are inherently contradictory. In this paper, we propose a resolution to this contradiction by demonstrating that the site of first antigen encounter dictates which mechanism of negative selection is utilized. We demonstrate that the bone marrow microenvironment provides signals that block antigen-induced deletion and promote RAG reinduction. In the periphery, the absence of these signals allows the immature B cell to default to apoptosis as a result of BCR engagement.

Tonic B cell antigen receptor signals supply an NF-κB substrate for prosurvival BLyS signaling

The survival of transitional and mature B cells requires both the B cell antigen receptor (BCR) and BLyS receptor 3 (BR3), which suggests that these receptors send signals that are nonredundant or that engage in crosstalk with each other. Here we show that BCR signaling induced production of the nonclassical transcription factor NF-κB pathway substrate p100, which is required for transmission of BR3 signals and thus B cell survival. The capacity for sustained p100 production emerged during transitional B cell differentiation, the stage at which BCR signals begin to mediate survival rather than negative selection. Our findings identify a molecular mechanism for the reliance of primary B cells on continuous BR3 and BCR signaling, as well as for the gradual resistance to negative selection that is acquired during B cell maturation.

Transitional B cells: step by step towards immune competence

Transitional B cells mark the crucial link between bone-marrow (BM) immature and peripheral mature B cells. Examination reveals unexpected heterogeneity, consisting of contiguous subsets with phenotypic and functional differences. Data point to the late transitional B-cell stage as a crucial juncture at which developing B cells gain access to splenic follicles, become responsive to T-cell help and lose sensitivity to negative selection, characterizing the immature B-cell response to B-cell antigen receptor (BCR) signaling in vitro and in vivo. The biological and molecular processes directing maturation through this stage are becoming clearer through biochemical studies and murine models deficient in various components of the BCR signaling pathway.

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. J. Leukoc. Biol. 60: 159–166; 1996.

Role of primary response genes in generating cellular responses to growth factors.

Cellular responses to growth and differentiation factors involve a complex cascade of signals that begins at the cell surface, traverses the cytoplasm, and eventually enters the nucleus. Although a great deal is known about the surface and cytoplasmic stages of this cascade, the nuclear events involved in transducing and translating growth and differentiation signals are less well understood. Recent work has implicated a set of genes known as primary response genes as critical for this process. To propagate the activation signal, these genes possess the ability not only to directly respond to upstream biochemical events, but also to transmit the signal downstream by modulating the unique changes in gene expression necessary for a particular cellular response. In this review we discuss how transcription factors encoded by primary response genes may be responsible for regulating tissue‐ or stimulus‐specific responses associated with cellular activation.— McMahon, S. B.; Monroe, J. G. Role of primary response genes in generating cellular responses to growth factors. FASEB J. 6: 2707‐2715; 1992.

Fate decisions regulating bone marrow and peripheral B lymphocyte development.

In adult mammals, bone marrow pluripotent hematopoietic stem cells generate B lymphoid-specified progeny that progress through a series of well-characterized stages before generating B-cell receptor expressing B lymphocytes. These functionally immature B lymphocytes then migrate to the spleen wherein they differentiate through transitional stages into follicular or marginal zone B lymphocytes capable of responding to T-dependent and -independent antigens, respectively. During the terminal stages of B lymphocyte development in the bone marrow, as well as immediately following egress into the peripheral compartments, B lymphocytes are counterselected to eliminate B lymphocytes with potentially dangerous self-reactivity. These developmental and selection events in the bone marrow and periphery are dependent on the integration of intrinsic genetic programs with extrinsic microenvironmental signals that drive progenitors toward increasing B lineage commitment and maturation. This chapter provides a comprehensive overview of the various stages of primary and secondary B lymphocyte development with an emphasis on the selection processes that affect decisions at critical checkpoints. Our intent is to stress the concept that at many steps in the developmental process leading to a mature immunocompetent B lymphocyte, B lineage cells are integrating multiple and different signaling inputs that are translated into specific and appropriate cell fate decisions.

MMTV Env encodes an ITAM responsible for transformation of mammary epithelial cells in three-dimensional culture

Expression of immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling proteins is normally restricted to hematopoietic tissues. The basal activity of ITAM-containing proteins is mediated through negative regulation by coreceptors restricted to hematopoietic tissues. We have identified an ITAM signaling domain encoded within the env gene of murine mammary tumor virus (MMTV). Three-dimensional structures derived in vitro from murine cells stably transfected with MMTV env display a depolarized morphology in comparison with control mammary epithelial cells. This effect is abolished by Y>F substitution within the Env ITAM, as well as inhibitors of Syk and Src protein tyrosine kinases. Env-expressing cells bear hallmarks of cell transformation such as sensitivity to apoptosis induced by tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) or TNFα, as well as down-regulation of E-cadherin and Keratin-18. Human normal mammary epithelial cells expressing MMTV Env also develop transformed phenotype, as typified by growth in soft agar and Matrigel invasion. These disruptions are abrogated by Y>F substitutions. We conclude that ITAM-dependent signals are generated through MMTV Env and trigger early hallmarks of transformation of mouse and human mammary epithelial cells. Therefore, these data suggest a heretofore unappreciated potential mechanism for the initiation of breast cancer and identify MMTV Env and ITAM-containing proteins in human breast tumors as probable oncoproteins.

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.

Lymphokine regulation of inflammatory processes: Interleukin-4 stimulates fibroblast proliferation

While recent evidence from several laboratories has shown that interleukin-4 (IL-4) can act on a number of cells in addition to B lymphocytes, these have thus far been limited to cells of the hematopoietic lineage. Here we report that murine IL-4 promotes DNA synthesis in both primary and immortalized fibroblasts. Marked stimulation of [3H]thymidine incorporation was observed for primary skin fibroblasts or Balb/c3T3 cells stimulated with HPLC- or immunoaffinity-purified as well as recombinant IL-4. Responses to immunoaffinity and recombinant IL-4 were completely blocked with anti-IL-4 antibody. Similar dose/response relationships were observed for recombinant IL-4 on skin fibroblasts and an IL-4 responsive murine T cell tumor, suggesting that the receptors for this lymphokine on these cells is similar. Together, these results show that IL-4 can cause DNA synthesis by murine fibroblasts presumably through ligand-receptor interactions at the cell surface. Implications of these findings to inflammation during an immune response is discussed.