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 repressor 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.

Mitogen plus interleukin 4 induction of C epsilon transcripts in B lymphoid cells.

To elucidate the mechanism of IL-4-induced enhancement of IgE and IgG1 production, murine splenic B cells and A-MuLV-transformed cells were cultured with LPS and IL-4 and assayed for epsilon and gamma 1 transcripts. Concomitant treatment with IL-4 and LPS induced expression of C epsilon transcripts in both normal and transformed cells. Expression of these truncated C epsilon transcripts preceded accumulation of normal epsilon mRNA in treated cells. Consistent data were obtained with respect to gamma 1 RNA expression. These results suggest that IL-4 can direct class switching in the context of a mechanism associated with differential expression of germline constant region genes.

The thrombopoietin receptor can mediate proliferation without activation of the Jak-STAT pathway.

Cytokine receptors of the hematopoietic receptor superfamily lack intrinsic tyrosine kinase domains for the intracellular transmission of their signals. Instead all members of this family associate with Jak family nonreceptor tyrosine kinases. Upon ligand stimulation of the receptors, Jaks are activated to phosphorylate target substrates. These include STAT (signal transducers and activators of transcription) proteins, which after phosphorylation translocate to the nucleus and modulate gene expression. The exact role of the Jak-STAT pathway in conveying growth and differentiation signals remains unclear. Here we describe a deletion mutant of the thrombopoietin receptor (c-mpl) that has completely lost the capacity to activate Jaks and STATs but retains its ability to induce proliferation. This mutant still mediates TPO-induced phosphorylation of Shc, Vav, mitogen-activated protein kinase (MAPK) and Raf-1 as well as induction of c-fos and c-myc, although at somewhat reduced levels. Furthermore, we show that both wild-type and mutant receptors activate phosphatidylinositol (PI) 3-kinase upon thrombopoietin stimulation and that thrombopoietin-induced proliferation is inhibited in the presence of the PI 3-kinase inhibitor wortmannin. These results demonstrate that the Jak-STAT pathway is dispensable for the generation of mitogenic signals by a cytokine receptor.

Transforming growth factor beta 1 selectivity stimulates immunoglobulin G2b secretion by lipopolysaccharide-activated murine B cells.

Bacterial lipopolysaccharide (LPS) has been reported to induce immunoglobulin (Ig)G2b class switching, yet we observed strain differences in IgG2b secretion in response to this mitogen. Specifically, BALB/c B cells, unlike those from DBA/2, synthesized relatively low amounts of IgG2b relative to IgG3, IgG1, or IgM. This report demonstrates that transforming growth factor (TGF) beta 1, previously shown to induce IgA class switching, selectively stimulates IgG2b secretion by BALB/c resting B cells activated with LPS. This activity was specifically reversed with a neutralizing anti-TGF-beta 1 antibody. The ability of TGF-beta 1 to act directly on highly purified membrane (m)IgM+ mIgG2b- cells to stimulate IgG2b production, stimulate an increase in IgG2b-secreting cells, and selectively increase the steady-state levels of germline gamma 2b RNA, suggests that it promotes IgG2b class switching. In this regard, addition of anti-TGF-beta antibody to cultures of DBA/2-derived resting B cells activated by LPS, alone, led to selective reduction in IgG2b secretion, indicating that endogenous TGF-beta 1 accounts for the high IgG2b secretory response observed in that strain. Finally, TGF-beta 1 failed to stimulate IgG2b secretion by B cells activated with dextran-conjugated anti-IgD antibody. We propose that TGF-beta 1 is a switch factor for the murine IgG2b subclass for appropriately activated B cells. In combination with other data, this would show that all six non-IgM, non-IgD isotypes in the mouse can be selectively induced by specific cytokines.

Interferon gamma signaling alters the function of T helper type 1 cells.

One mechanism regulating the ability of different subsets of T helper (Th) cells to respond to cytokines is the differential expression of cytokine receptors. For example, Th2 cells express both chains of the interferon gamma receptor (IFN-gammaR), whereas Th1 cells do not express the second chain of the IFN-gammaR (IFN-gammaR2) and are therefore unresponsive to IFN-gamma. To determine whether the regulation of IFN-gammaR2 expression, and therefore IFN-gamma responsiveness, is important for the differentiation of naive CD4(+) T cells into Th1 cells or for Th1 effector function, we generated mice in which transgenic (TG) expression of IFN-gammaR2 is controlled by the CD2 promoter and enhancer. CD4(+) T cells from IFN-gammaR2 TG mice exhibit impaired Th1 polarization potential in vitro. TG mice also display several defects in Th1-dependent immunity in vivo, including attenuated delayed-type hypersensitivity responses and decreased antigen-specific IFN-gamma production. In addition, TG mice mount impaired Th1 responses against Leishmania major, as manifested by increased parasitemia and more severe lesions than their wild-type littermates. Together, these data suggest that the sustained expression of IFN-gammaR2 inhibits Th1 differentiation and function. Therefore, the acquisition of an IFN-gamma-unresponsive phenotype in Th1 cells plays a crucial role in the development and function of these cells.

Jak-STAT signaling induced by the v-abl oncogene.

The effect of the v-abl oncogene of the Abelson murine leukemia virus (A-MuLV) on the Jak-STAT pathway of cytokine signal transduction was investigated. In murine pre-B lymphocytes transformed with A-MuLV, the Janus kinases (Jaks) Jak1 and Jak3 exhibited constitutive tyrosine kinase activity, and the STAT proteins (signal transducers and activators of transcription) normally activated by interleukin-4 and interleukin-7 were tyrosine-phosphorylated in the absence of these cytokines. Coimmunoprecipitation experiments revealed that in these cells v-Abl was physically associated with Jak1 and Jak3. Inactivation of v-Abl tyrosine kinase in a pre-B cell line transformed with a temperature-sensitive mutant of v-abl resulted in abrogation of constitutive Jak-STAT signaling. A direct link may exist between transformation by v-abl and cytokine signal transduction.

Lack of interferon gamma receptor beta chain and the prevention of interferon gamma signaling in TH1 cells.

The ability of interferon gamma (IFN-gamma) to inhibit the proliferation of type 2 T helper cells (TH2), but not that of type 1 (TH1) cells, suggests that helper cell subsets might differ in their activation of the IFN-gamma signaling pathway. The IFN-gamma-inducible signal transducing factor (STF-IFN gamma) was activated in murine TH2 but not in TH1 cell clones, because in the latter the second chain of the IFN-gamma receptor (accessory factor 1 or IFN-gamma R beta) was absent. Thus, TH1 cells use receptor modification to prevent the activation of STF-IFN gamma and achieve an IFN-gamma-resistant state.

IL-4 receptor mutations.

IL-4 plays an important role in regulating immune responses. Distinct signaling pathways, including those for gene activation and cell differentiation and those for cell proliferation and protection from apoptosis, are initiated from the receptor complex for IL-4 following ligand-receptor engagement. Several advances have been made in our understanding of how distinct functions of IL-4 are mediated. Most of these studies employed artificial mutations of the IL-4-receptor alpha chain using site-directed mutagenesis and/or deletional mutation. In addition, naturally occurring mutations of the IL-4-receptor alpha chain have been identified and implicated as a genetic predisposition for allergic disorders. The results of these studies suggest a modular organization of the receptor and an independent regulation of gene activation and cell growth.

Identification of a conserved lipopolysaccharide-plus-interleukin-4-responsive element located at the promoter of germ line epsilon transcripts.

Treatment of splenic B lymphocytes and certain B-lineage cell lines with the mitogen lipopolysaccharide (LPS) and the lymphokine interleukin-4 (IL-4) induces expression of germ line immunoglobulin C epsilon transcripts and class switching to the C epsilon gene. We show that LPS-plus-IL-4 induction of germ line epsilon transcripts (termed I epsilon transcripts) occurs at the transcriptional level in an Abelson murine leukemia virus-transformed pre-B-cell line. A 1.1-kb region of DNA surrounding the I epsilon promoter endows inducible transcription to a heterologous reporter gene stably transfected into these cells; such inducible expression depends on combined treatment with LPS and IL-4. Analyses of constructs transiently introduced into a B-cell lymphoma line demonstrated that LPS-plus-IL-4-inducible expression can be conferred by a 179-bp segment of DNA spanning the I epsilon transcriptional initiation site. Mutational analyses demonstrated that this expression depended on DNA sequences within a conserved region directly upstream from the I epsilon transcriptional initiation region. One nuclear protein that is constitutively expressed in normal B cells binds to the downstream end of the conserved sequence; its binding specificity correlates with the functional effect of several mutations. Two additional proteins, which are induced by IL-4 treatment of splenic B cells, bind to the transcription initiation sites of I epsilon. These proteins are indistinguishable in binding assays from proteins previously shown to bind an enhancer region of the class II major histocompatibility complex gene A alpha.

Structure and expression of germ line immunoglobulin heavy-chain epsilon transcripts: interleukin-4 plus lipopolysaccharide-directed switching to C epsilon.

We have isolated cDNA clones complementary to a truncated immunoglobulin heavy-chain C epsilon RNA transcript previously found to be induced in B lymphoid cells by treatment with lipopolysaccharide (LPS) combined with interleukin-4 (IL-4). We demonstrate that this transcript initiates from a promoter upstream of the germ line epsilon class-switch recombination region (S epsilon region). The major germ line C epsilon transcript contains a small 5' exon contributed by sequences upstream of the S epsilon region spliced to the normal C epsilon exons. Treatment of splenic B lymphoid cells with LPS plus IL-4 induces the expression of transcripts from the germ line epsilon transcription unit followed by expression of normal immunoglobulin epsilon heavy-chain mRNA. Furthermore, we demonstrate that similar treatment of transformed precursor B cell lines induces the expression of germ line epsilon transcripts followed by class switching to epsilon expression in these lines. This is the first demonstration of switching to epsilon in cells of the pre-B stage. The general structure of the germ line epsilon transcript and transcription unit is similar to that previously characterized for germ line gamma 2b transcripts. However, expression of these two germ line transcription units in B-lineage cells is inversely regulated by IL-4 (plus LPS) treatment, correlating with the effects of these treatments on switching to these loci.

Direct interaction of Jak1 and v-Abl is required for v-Abl-induced activation of STATs and proliferation.

In Abelson murine leukemia virus (A-MuLV)-transformed cells, members of the Janus kinase (Jak) family of non-receptor tyrosine kinases and the signal transducers and activators of transcription (STAT) family of signaling proteins are constitutively activated. In these cells, the v-Abl oncoprotein and the Jak proteins physically associate. To define the molecular mechanism of constitutive Jak-STAT signaling in these cells, the functional significance of the v-Abl-Jak association was examined. Mapping the Jak1 interaction domain in v-Abl demonstrates that amino acids 858 to 1080 within the carboxyl-terminal region of v-Abl bind Jak1 through a direct interaction. A mutant of v-Abl lacking this region exhibits a significant defect in Jak1 binding in vivo, fails to activate Jak1 and STAT proteins, and does not support either the proliferation or the survival of BAF/3 cells in the absence of cytokine. Cells expressing this v-Abl mutant show extended latency and decreased frequency in generating tumors in nude mice. In addition, inducible expression of a kinase-inactive mutant of Jak1 protein inhibits the ability of v-Abl to activate STATs and to induce cytokine-independent proliferation, indicating that an active Jak1 is required for these v-Abl-induced signaling pathways in vivo. We propose that Jak1 is a mediator of v-Abl-induced STAT activation and v-Abl induced proliferation in BAF/3 cells, and may be important for efficient transformation of immature B cells by the v-abl oncogene.

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

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 epsilon 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 epsilon 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 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. Oncogene (2000).

Interferon-gamma receptor signaling is dispensable in the development of autoimmune type 1 diabetes in NOD mice.

There have been two previous conflicting reports that the development of T-cell-mediated autoimmune diabetes (type 1 diabetes) was respectively unaffected or inhibited in NOD mice genetically deficient in the T-helper (Th) 1 cytokine interferon (IFN)-gamma or the alpha-chain subunit of its receptor. Our goal was to resolve this conundrum by congenically transferring, from a 129 donor strain to the NOD background, a functionally inactivated gene for the beta-chain signaling (located on chromosome 16) rather than the alpha-chain ligand binding domain (located on chromosome 10) of the IFN-gamma receptor. These NOD.IFNgammaRBnull mice were characterized by normal patterns of leukocyte development and T-cells that produced greatly enhanced levels of the putatively type 1 diabetes-protective Th2 cytokine interleukin (IL)-4. However, despite being unable to respond to the primary Thl cytokine IFN-gamma and having T-cells that produce greatly enhanced levels of IL-4, NOD.IFNgammaRBnull mice remained highly susceptible to type 1 diabetes. This result indicated that the previously reported inhibition of type 1 diabetes in NOD mice carrying a functionally inactivated IFN-gamma receptor alpha-chain gene may have been due to a closely linked and previously unidentified diabetes resistance allele. Furthermore, our results indicate that the pathogenicity of diabetogenic T-cells in NOD mice is not dampened by an inability to respond to IFN-gamma and a concurrent shift to greatly enhanced Th2 cytokine production. This finding calls into question whether clinical protocols designed to shift beta-cell autoreactive T-cells from a Thl to Th2 cytokine production profile will truly be safe and efficacious in blocking the development of type 1 diabetes in humans.

Isolation of cDNAs encoding T-BAM, a surface glycoprotein on CD4+ T cells mediating contact-dependent helper function for B cells: identity with the CD40-ligand.

"T-cell B-cell Activating Molecule" (T-BAM) is an activation-induced surface protein on CD4+ T cells that mediates a contact-dependent signal for B cell differentiation and immunoglobulin (Ig) secretion. The T-BAM protein on a helper clone of Jurkat (D1.1) was affinity purified using the anti-T-BAM mAb, 5c8. The NH2-terminal amino acid sequence of purified T-BAM was determined and found to be highly homologous to the predicted NH2-terminal sequence of a T cell ligand to the B cell CD40 molecule (CD40-L). From a D1.1 cDNA library, a clone was isolated that encodes CD40-L by sequence and drives expression of T-BAM protein on transfected cells, demonstrating that the T-BAM and CD40-L genes and proteins are identical. Moreover, transfection of T-BAM was shown to confer to non-lymphoid cells, the ability to induce B cells to upregulate the expression of surface CD23 molecules. In previous studies we showed that T-BAM was expressed predominantly on activated CD4+ and on few if any CD8+ cells. Although the current work confirms that T-BAM is largely restricted to activated CD4+ T cells, we now provide definitive evidence that T-BAM can be expressed by a small population of CD8+ T cells after activation. Importantly, a subset of CD8+ T cells do not express T-BAM after activation and this T-BAM- phenotype is maintained on certain CD8+ T cell clones. Taken together, these data unify the biology and structure of T-BAM and CD40-L and this synthesis has implications for understanding the T cell regulation of the humoral immune response.

Endocrine effects and pharmacokinetic characteristics of a potent new gonadotropin-releasing hormone antagonist (Ganirelix) with minimal histamine-releasing properties: studies in postmenopausal women.

A potent and safe GnRH antagonist has been sought unsuccessfully for the last 2 decades. The recently developed GnRH antagonist RS-26306 or Ganirelix ([N-Ac-D-Nal(2)1,D-pClPhe2,D-Pal(3)3,D-hArg(Et2)6,L-++ +hArg(Et2)8,D-Ala10]GnRH ; Syntex Research, Palo Alto, CA), exhibited high antiovulatory potency and low histamine-releasing properties in preclinical studies. Therefore, we determined the extent to which single sc injections of three doses of RS-26306 (1, 3, and 6 mg) decreased serum concentrations of LH and FSH, the free alpha-subunit of LH/FSH/TSH, PRL, and testosterone in five healthy postmenopausal women. We also examined the pharmacokinetic characteristics of RS-26306 by quantifying serum levels of the drug by RIA. RS-26306 rapidly suppressed serum concentrations of LH, FSH, and free alpha-subunit. RS-26306 (6 mg) maximally decreased serum concentrations (mean +/- SEM) of LH, FSH, and free alpha-subunit by 70.1 +/- 3.6%, 42.3 +/- 2.5%, and 74.6 +/- 3.5%, respectively. RS-26306 also decreased serum testosterone, but not serum PRL, concentrations. RS-26306 concentrations reached peak serum levels at 1.2 +/- 0.3, 1.9 +/- 0.4, and 1.8 +/- 0.5 h, respectively, after 1-, 3-, and 6-mg sc injections. The mean serum half-life values based on the terminal portion of the disappearance curves were 22.8 +/- 2.5 and 26.9 +/- 1.0 h, respectively, after 3- and 6-mg s.c. doses. No systemic side-effects were noted after the administration of RS-26306. Our results demonstrate that the GnRH antagonist RS-26306 has favorable pharmacokinetic characteristics and is a potent suppressor of pituitary gonadotropin secretion in postmenopausal women. These attributes and the lack of systemic side-effects make RS-26306 a promising candidate for future clinical applications.

Binding of trans-acting factors to the double-stranded variant surface glycoprotein (VSG) expression site promoter of Trypanosoma brucei.

Trypanosoma brucei evades its host's immune response by utilizing the system of antigenic variation, whereby the organism sequentially expresses antigenically distinct variant surface glycoproteins (VSGs). Actively expressed VSG genes are found in VSG expression sites (ESs), and transcription of these ESs is directed by a small promoter composed of two essential cis-acting elements, the VSG ES promoter upstream element (VUE) and VSG ES promoter downstream element (VDE). Using electrophoretic mobility shift assays, we have identified double-stranded DNA binding activity in bloodstream-form trypanosome nuclear extracts. This activity, the VEP complex, is specific for the VSG ES promoter, and requires the intact sequences of the VUE and VDE in the appropriate spacing. These requirements of VEP Complex formation parallel the requirements for promoter function, suggesting that the VEP complex may be composed of functionally significant trans-acting factors. Furthermore, the requirement of both elements suggests that the binding of factors to the promoter may be cooperative. However, subtly different binding characteristics were observed when we used nuclear extracts derived from procyclic trypanosomes.

Control of recombination events during lymphocyte differentiation. Heavy chain variable region gene assembly and heavy chain class switching.

Our recent studies have focused on the organization of immunoglobulin genes in mice and humans and the mechanism and control of the recombination events that are involved in their assembly and expression. This report describes our progress in this area with particular focus on elucidating factors that influence the generation of the antibody repertoire in normal and diseased states. We present a detailed analysis of the organization of the human VH locus, studies that help to elucidate the nature of the recombination defect in mice with severe combined immunodeficiency, and studies of transgenic mice that focus on the mechanism that regulates tissue-specific variable region gene assembly. In addition, we also characterize mechanisms that control the heavy chain class-switch process. Although the latter process apparently involve a recombination system distinct from that involved in variable region assembly, we find that the two recombination events appear to be controlled by similar mechanisms.

Amniotic membranes in the treatment of necrotizing fasciitis complicating vulvar herpes virus infection.

Necrotizing fasciitis of the vulva developed in an immunocompromised patient with chronic myelogenous leukemia, apparently from secondarily infected herpes simplex lesions. In addition to surgical debridements and broad-spectrum intravenous antibiotic therapy, the wound was treated using specially prepared amniotic membranes as a wound dressing. The patient died on hospital day 65 because of complications of her immunocompromised state, with autopsy findings of disseminated cytomegalovirus. However, use of amniotic membranes as a wound dressing appeared to be beneficial.

Urethral T-cell lymphoma as the initial manifestation of the acquired immune deficiency syndrome.

We report the first case (to our knowledge) of a primary urethral T-cell lymphoma as the initial manifestation of the acquired immune deficiency syndrome. A 36-year-old white homosexual man with antibodies to human immunodeficiency virus type 1 was evaluated for a hemorrhagic urethral discharge. A 2-cm fleshy, polypoid mass in the bulbous urethra was removed, and the diagnosis of small non-cleaved cell (non-Burkitt's) lymphoma was made. Immunohistochemical analysis confirmed that the tumor was of T-cell lineage. Patients with the acquired immunodeficiency syndrome have an increased incidence of lymphomas, particularly extranodal high-grade non-Hodgkin's lymphomas. Primary urethral lymphomas are extremely rare, with only a handful of cases reported in the literature. This rare form and site of lymphoma should be considered in patients with the acquired immunodeficiency syndrome who have genitourinary symptoms.