Paul Rothman

The BCL-6 proto-oncogene controls germinal-centre formation and Th2- type inflammation

Structural alterations of the promoter region of the BCL-6 proto-oncogene represent the most frequent genetic alteration associated with non-Hodgkin lymphoma, a malignancy often deriving from germinal-centre B cells. The BCL-6 gene encodes a zinc-finger transcriptional represser normally expressed in both B cells and CD4+ T cells within germinal centres, but its precise function is unknown. We show that mice deficient in BCL-6 displayed normal B-cell, T-cell and lymphoid-organ development but have a selective defect in T-cell-dependent antibody responses. This defect included a complete lack of affinity maturation and was due to the inability of follicular B cells to proliferate and form germinal centres. In addition, BCL-6-deficient mice developed an inflammatory response in multiple organs characterized by infiltrations of eosinophils and IgE-bearing B lymphocytes typical of a Th2-mediated hyperimmune response. Thus, BCL-6 functions as a transcriptional switch that controls germinal centre formation and may also modulate specific T-cell-mediated responses. Altered expression of BCL-6 in lymphoma represents a deregulation of the pathway normally leading to B cell proliferation and germinal centre formation.

Mechanism and regulation of immunoglobulin isotype switching.

Publisher Summary This chapter focuses on the molecular mechanisms of switch recombination and other possible modes of isotype switching. It discusses the regulation of isotype switching, especially by T cells and the cytokines they produce. Virtually all of the analyses of the mechanisms and regulation of switching are done in either mouse or human, with a disproportionate amount done in the mouse. The two species are discussed separately, although the basic lessons learned from the mouse are, for the most part, valid for humans as well. Isotype switching could be induced by the direct actions of mitogens or antigens on B cells, as shown by the ability of a variety of T-independent antigens and mitogens to induce immunoglobulin (IgG), especially IgG3, in mice. Isotype switching involves an intrachromosomal recombination that brings the fully assembled and expressed heavy chain variable (V H ) gene into close proximity to a new C H gene, with the resulting deletion of Cμ Cδ, and any other intervening C H genes. The consequence of this essentially irreversible cellular differentiation is the production of antibody molecules that retain the same antigen specificity, but connect it with different effector functions.

S region transcription per se promotes basal IgE class switch recombination but additional factors regulate the efficiency of the process

Stimulation of B lymphocytes with a combination of lipopolysaccharide (LPS) and interleukin‐4 (IL‐4) induces germline transcription of and subsequent switching to the epsilon heavy chain constant region (C epsilon) gene. Mature germline C epsilon transcripts contain a non‐coding exon (I epsilon exon) spliced to the C epsilon exons. To distinguish between the potential roles of germline transcription and those of germline transcripts in regulating the class switch process, we replaced the LPS‐ and IL‐4‐inducible I epsilon promoter and exon in ES cells with an LPS‐inducible E mu enhancer/VH promoter expression cassette. Wildtype, heterozygous or homozygous mutant ES cells were injected into RAG‐2 deficient blastocysts to generate somatic chimeras in which all B cells derived from ES cells. In contrast to normal B cells, heterozygous and homozygous mutant B cells had substantial transcription through the epsilon switch recombination region (S epsilon) following treatment with LPS alone and, under these conditions, both underwent low level switching (10‐ to 100‐fold less than wildtype cells stimulated with LPS + IL‐4) to IgE production. Heterozygous mutant cells underwent switching to IgE at essentially wildtype levels when stimulated with LPS and IL‐4. However, homozygous mutant cells still showed extremely low levels of switching to IgE upon LPS and IL‐4 stimulation. Analyses of hybridomas from heterozygous mutants indicated that the mutation is cis‐acting and normal switching to other isotypes indicated that it is specific for IgE. Thus transcription per se generates low levels of class switch recombination in the absence of I region sequences. However, we demonstrate for the first time that, for optimal efficiency, the process requires the presence of the intact I region and/or I region promoter in cis, implicating factors beyond transcription through the S region in the regulation of class switching.

Pim serine/threonine kinases regulate the stability of Socs-1 protein

Studies of SOCS-1-deficient mice have implicated Socs-1 in the suppression of JAK-STAT (Janus tyrosine kinase-signal transducers and activators of transcription) signaling and T cell development. It has been suggested that the levels of Socs-1 protein may be regulated through the proteasome pathway. Here we show that Socs-1 interacts with members of the Pim family of serine/threonine kinases in thymocytes. Coexpression of the Pim kinases with Socs-1 results in phosphorylation and stabilization of the Socs-1 protein. The protein levels of Socs-1 are significantly reduced in the Pim-1−/−, Pim-2−/− mice as compared with wild-type mice. Similar to Socs-1−/− mice, thymocytes from Pim-1−/−, Pim-2−/− mice showed prolonged Stat6 phosphorylation upon IL-4 stimulation. These data suggest that the Pim kinases may regulate cytokine-induced JAK-STAT signaling through modulation of Socs-1 protein levels.

STF-IL-4: a novel IL-4-induced signal transducing factor.

The mechanism by which interleukin‐4 (IL‐4) regulates the expression of particular genes is unknown. We have determined that IL‐4 induces a DNA binding factor (termed STF‐IL‐4) which has a strong affinity for an IFN‐gamma activation site (GAS). Interestingly, STF‐IL‐4 also binds to the IL‐4 responsive promoter for the Ig heavy chain germline epsilon transcript. The IL‐4 dependent activation of STF‐IL‐4 is rapid, does not require protein synthesis and results in the sequential appearance of binding activity first in the cytoplasm and then later in the nucleus. Activation of STF‐IL‐4 is sensitive to tyrosine kinase inhibitors and the active factor is tyrosine phosphorylated. This pattern of activation is similar to the activation of interferon‐induced transcription factors. STF‐IL‐4 appears to be a new member of a growing family of cytokine‐induced transcriptional regulators.

Transcriptional repression of Stat6-dependent interleukin-4-induced genes by BCL-6: specific regulation of iepsilon transcription and immunoglobulin E switching.

ABSTRACT The BCL-6 proto-oncogene encodes a POZ/zinc-finger transcription factor that is expressed in B cells and a subset of CD4+ T cells within germinal centers. Recent evidence suggests that BCL-6 can act as a sequence-specific repressor of transcription, but the target genes for this activity have not yet been identified. The binding site for BCL-6 shares striking homology to the sites that are the target sequence for the interleukin-4 (IL-4)-induced Stat6 (signal transducers and activators of transcription) signaling molecule. Electrophoretic mobility shift assays demonstrate that BCL-6 can bind, with different affinities, to several DNA elements recognized by Stat6. Expression of BCL-6 can repress the IL-4-dependent induction of immunoglobulin (Ig) germ line ɛ transcripts, but does not repress the IL-4 induction of CD23 transcripts. Consistent with the role of BCL-6 in modulating transcription from the germ line ɛ promoter, BCL-6−/−mice display an increased ability to class switch to IgE in response to IL-4 in vitro. These animals also exhibit a multiorgan inflammatory disease characterized by the presence of a large number of IgE+ B cells. The apparent dysregulation of IgE production is abolished in BCL-6−/− Stat6−/− mice, indicating that BCL-6 regulation of Ig class switching is dependent upon Stat6 signaling. Thus, BCL-6 can modulate the transcription of selective Stat6-dependent IL-4 responses, including IgE class switching in B cells.

JAK-STAT signaling in asthma

The past two decades have witnessed a dramatic increase in the prevalence of asthma worldwide (1). Asthma is a chronic disease characterized by variable airway obstruction, airway hyperresponsiveness (AHR), and airway inflammation and remodeling. Histological studies show that airways of asthmatic patients contain a chronic inflammatory infiltrate composed of lymphocytes, eosinophils, and mast cells. This infiltrate is usually accompanied by desquamation of the bronchial epithelial layer, goblet cell hyperplasia, and thickening of the submucosa. In most cases, the asthmatic inflammatory process results from inappropriate immune responses to common environmental antigens in a genetically susceptible individual (2). These inappropriate immune responses are orchestrated by a subset of CD4+ T helper cells termed T helper 2 (Th2) cells. Cytokines play a pivotal role in the development of asthma by regulating the expansion of Th2 cells and by mediating many of the Th2 effector functions that underlie the pathogenic events of an asthmatic response. Much effort has recently been placed in elucidating the pathways used by cytokines to mediate their actions. These studies have revealed that cytokine-mediated signals are primarily transduced by the Jak-Stat signaling cascade (3). In this review we will highlight the recent advances made in dissecting the roles of this signaling pathway in the pathogenesis of asthma.

Growth and Gene Expression Are Predominantly Controlled by Distinct Regions of the Human IL-4 Receptor

IL-4 causes hematopoietic cells to proliferate and express a series of genes, including CD23. We examined whether IL-4-mediated growth, as measured by 4PS phosphorylation, and gene induction were similarly controlled. Studies of M12.4.1 cells expressing human IL-4R truncation mutants indicated that the region between amino acids 557-657 is necessary for full gene expression, which correlated with Stat6 DNA binding activity. This region was not required for 4PS phosphorylation. Tyrosine-to-phenylalanine mutations in the interval between amino acids 557-657 revealed that as long as one tyrosine remained unmutated, CD23 was fully induced. When all three tyrosines were mutated, the receptor was unable to induce CD23. The results indicate that growth regulation and gene expression are principally controlled by distinct regions of IL-4R.

SOCS Proteins, Regulators of Intracellular Signaling

The mechanism by which cytokine signaling is regulated has been a focus of research in the past few years. The SOCS family proteins have recently emerged as important regulators of cytokine signaling. Although the first two family members, CIS and SOCS-1, were identified as cytokine-inducible inhibitors of JAK-STAT activation, it is not clear whether other family members also act similarly. Gene disruption experiments have suggested a critical role for SOCS-1 in the regulation of IFNγ signaling and T cell development. SOCS-2 and SOCS-3 have also been shown to be important in postnatal growth and in fetal liver erythropoiesis, respectively. The precise mechanisms by which these proteins exert their effects remain to be determined. In addition, the function of the conserved SOCS box will be another focus of research. Although the SOCS box has been proposed to be involved in the ubiquitylation of proteins, there has been no direct evidence supporting a role for SOCS proteins in cellular ubiquitylation. Overall, identification of the SOCS family has opened a new field of research in signal transduction. Studies of these proteins will not only increase our understanding of immune regulation by the cytokine network but may also lead to development of specific pharmacological inhibitors of cytokine signaling.*E-mail: pbr3@columbia.edu.

JAK-STAT signaling activated by Abl oncogenes

The Abl oncoproteins v-Abl and BCR-Abl can activate member of the signal transducers and activators of transcription (STAT) family of signaling proteins. The mechanisms by which these oncoproteins activate STATs appear to differ. In cells transformed by v-Abl, Janus kinase (JAK) tyrosine kinases are constitutively activated. In these cells, the v-Abl oncoprotein and the JAK kinases physically associate. Mapping of the JAK interaction domain in v-Abl demonstrates that amino acids within the carboxyl terminal region of v-Abl bind JAKs through a direct interaction. A mutant of v-Abl lacking this region does not bind or activate JAK 1 in vivo, fails to activate STAT proteins, does not induce cellular proliferation, and is less efficient in cellular transformation. Kinase inactive mutants of JAK 1 inhibit the ability of v-Abl to activate STATs, to induce cytokine-independent proliferation, and to transform bone marrow cells. Interestingly, these effects correlate with defects in the activation of several pathways by v-Abl including Akt, PI3-kinase, STATs, and Ras. These data suggest that Jak kinases may play an important role in v-Abl induced transformation.