CREB-binding protein (CBP) regulates ß-adrenoceptor (ß-AR)-mediated apoptosis.

Catecholamines regulate the ß-adrenoceptor/cyclic AMP-regulated protein kinase A (cAMP/PKA) pathway. Deregulation of this pathway can cause apoptotic cell death and is implicated in a range of human diseases, such as neuronal loss during aging, cardiomyopathy and septic shock. The molecular mechanism of this process is, however, only poorly understood. Here we demonstrate that the ß-adrenoceptor/cAMP/PKA pathway triggers apoptosis through the transcriptional induction of the pro-apoptotic BH3-only Bcl-2 family member Bim in tissues such as the thymus and the heart. In these cell types, the catecholamine-mediated apoptosis is abrogated by loss of Bim. Induction of Bim is driven by the transcriptional co-activator CBP (CREB-binding protein) together with the proto-oncogene c-Myc. Association of CBP with c-Myc leads to altered histone acetylation and methylation pattern at the Bim promoter site. Our findings have implications for understanding pathophysiology associated with a deregulated neuroendocrine system and for developing novel therapeutic strategies for these diseases.

Complex regulation of cell-cycle inhibitors by Fbxw7 in mouse embryonic fibroblasts.

The F-box protein Fbxw7 (also known as Fbw7, SEL-10, hCdc4 or hAgo) mediates the ubiquitylation and thereby contributes to the degradation of proteins that positively regulate cell cycle. Conditional ablation of Fbxw7 in mouse embryonic fibroblasts (MEFs) induces cell-cycle arrest accompanied by abnormal accumulation of the intracellular domain of Notch1 (NICD1) and c-Myc. However, the molecular mechanisms by which the accumulation of NICD1 and c-Myc induces cell-cycle arrest have remained unclear. We have now examined the expression of cell-cycle inhibitors in Fbxw7-deficient MEFs and found that the abundance of p27(Kip1) and p57(Kip2) is paradoxically decreased. This phenomenon appears to be attributable to the accumulation of NICD1, given that it was recapitulated by overexpression of NICD1 and blocked by ablation of RBP-J. Conversely, the expression of p16(Ink4a) and p19(ARF) was increased in an NICD1-independent manner in Fbxw7-null MEFs. The increased expression of p19(ARF) was recapitulated by overexpression of c-Myc and abolished by ablation of c-Myc, suggesting that the accumulation of c-Myc is primarily responsible for that of p19(ARF). In contrast, the upregulation of p16(Ink4a) appeared to be independent of c-Myc. These results indicate that cell-cycle inhibitors undergo complex regulation by the Fbxw7-mediated proteolytic system.

c-Myc-deficient B lymphocytes are resistant to spontaneous and induced cell death.

C-myc gene is a member of the myc family of proto-oncogenes involved in proliferation, differentiation, and apoptosis. Overexpression of c-myc in fibroblasts causes apoptosis under low serum conditions in a process that requires the interaction of CD95 and CD95L on the surface. We have previously reported an in vivo conditional model to inactivate the c-myc gene in B lymphocytes. Here, we show that c-Myc-deficient primary B lymphocytes are resistant to different apoptotic stimuli. Nonactivated c-Myc-deficient B cells are resistant to spontaneous cell death. Upon activation, c-Myc-deficient B lymphocytes express normal surface levels of activation markers, and show resistance to staurosporine and CD95-induced cell death.

Analysis of C-MYC function in normal cells via conditional gene-targeted mutation.

Germline inactivation of c-myc in mice causes embryonic lethality. Therefore, we developed a LoxP/Cre-based conditional mutation approach to test the role of c-myc in mouse embryonic fibroblasts (MEFs) and mature B lymphocytes. Cre expression resulted in reduced proliferation of wild-type MEFs, but c-Myc-deficient MEFs showed a further reduction. In contrast to fibroblasts, Cre expression had no apparent affect on wild-type B cell proliferation. Deletion of both c-Myc genes in B cells led to severely impaired proliferation in response to anti-CD40 plus IL-4. However, treated cells did upregulate several early activation markers but not CD95 or CD95 ligand. We discuss these findings with respect to potential c-Myc functions in proliferation and apoptosis and also discuss potential limitations in the Cre-mediated gene inactivation approach.

Myc-enhanced expression of Cul1 promotes ubiquitin-dependent proteolysis and cell cycle progression.

The c-Myc oncoprotein plays an important role in the growth and proliferation of normal and neoplastic cells. To execute these actions, c-Myc is thought to regulate functionally diverse sets of genes that directly govern cellular mass and progression through critical cell cycle transitions. Here, we provide several lines of evidence that c-Myc promotes ubiquitin-dependent proteolysis by directly activating expression of the Cul1 gene, encoding a critical component of the ubiquitin ligase SCF(SKP2). The cell cycle inhibitor p27(kip1) is a known target of the SCF(SKP2) complex, and Myc-induced Cul1 expression matched well with the kinetics of declining p27(kip1) protein. Enforced Cul1 expression or antisense neutralization of p27(kip1) was capable of overcoming the slow-growth phenotype of c-Myc null primary mouse embryonic fibroblasts (MEFs). In reconstitution assays, the addition of in vitro translated Cul1 protein alone was able to restore p27(kip1) ubiquitination and degradation in lysates derived from c-myc(-/-) MEFs or density-arrested human fibroblasts. These functional and biochemical data provide a direct link between c-Myc transcriptional regulation and ubiquitin-mediated proteolysis and together support the view that c-Myc promotes G(1) exit in part via Cul1-dependent ubiquitination and degradation of the CDK inhibitor, p27(kip1).

N-myc can functionally replace c-myc in murine development, cellular growth, and differentiation.

Members of the myc family of cellular oncogenes have been implicated as transcriptional regulators in pathways that govern cellular proliferation and death. In addition, N-myc and c-myc are essential for completion of murine embryonic development. However, the basis for the evolutionary conservation of myc gene family has remained unclear. To elucidate this issue, we have generated mice in which the endogenous c-myc coding sequences have been replaced with N-myc coding sequences. Strikingly, mice homozygous for this replacement mutation can survive into adulthood and reproduce. Moreover, when expressed from the c-myc locus, N-myc is similarly regulated and functionally complementary to c-myc in the context of various cellular growth and differentiation processes. Therefore, the myc gene family must have evolved, to a large extent, to facilitate differential patterns of expression.

Molecular analysis of HIV-1 gp120 antibody response using isotype IgM and IgG phage display libraries from a long-term non-progressor HIV-1-infected individual.

To characterize the variable heavy chain (VH)3 antibody response to HIV-1 gp120, we analyzed a panel of IgM and IgG1 Fab fragments from phage display isotype libraries from a long-term, non-progressor HIV-1-infected individual. The IgM Fab antibodies isolated had low affinity for gp120, were not restricted to a particular VH3 germ-line gene, and consisted mainly of unmutated VH genes. In contrast, IgG Fab fragments were gp120 specific, with high affinity and extensive somatic mutation; all were clonally related and were derived from a single VH3 germ-line gene (DP50). One IgG Fab (S8) has DP50 VH region nucleotide substitutions identical to those of IgM Fab M025 and uses similar DH and JH segments, suggesting that S8 arose from M025 by isotype switching. In addition, somatic mutation in the IgG heavy chain third complementarity-determining region results in a 100-fold affinity increase for gp120, which correlates with a similar increase in neutralization capacity. These results imply that in vivo IgM to IgG isotype switch and affinity maturation may be important for protection and long-term survival in certain HIV-1-infected individuals.

Natural human antibodies retrieved by phage display libraries from healthy donors: polyreactivity and recognition of human immunodeficiency virus type 1gp120 epitopes.

We have characterized the human natural antibody repertoire that contains antibodies recognizing the human immunodeficiency virus type 1 (HIV-1) gp120. A panel of monovalent antigen-binding fragments (Fab) selected from IgM and IgG isotype libraries generated from peripheral blood mononuclear cells (PBMC) of a healthy, HIV-1 noninfected individual was analysed, reflecting that only IgM, but not IgG, Fab were able to recognize HIV-1 gp120. The IgM Fab antibodies were not restricted to any particular heavy chain variable region (VH) germ line gene. However, the recognition of gp120 is associated to polyreactive antibodies and all display low-affinity interaction. This correlates with the absence of any maturation process as somatic mutation or isotype switch as the nucleotide sequence analysis of the variable regions reveals they are expressed near to germline configuration. In addition, none of the antibodies showed any neutralizing activity on HIV-1-infected lymphocytes, reflecting that the natural anti-gp120 repertoire is not sufficient to neutralize HIV infection.

Human monoclonal Fab fragments specific for viral antigens from combinatorial IgA libraries.

BACKGROUND: IgA constitutes the first line of immune defense, interacting with a variety of environmental antigens. Following infection with respiratory syncytial virus (RSV) individuals frequently exhibit elevated serum IgA titers specific for the virus. Previously combinatorial IgG libraries have successfully been used to clone such human antibody responses. OBJECTIVES: Here we evaluate the possibility of constructing combinatorial IgA libraries on the surface of filamentous phage to retrieve human viral-specific IgA Fab fragments. STUDY DESIGN: Bone marrow from an HIV-1 seropositive donor was used as RNA source to construct combinatorial IgA kappa and lambda libraries of approximately 10(7) clones. RESULTS: By affinity selection using an immobilized recombinant RSV FG protein, two unique IgA Fab fragments producing clones (AD5 and AD23) reactive with the selecting antigen were isolated. One of the Fab fragments was found to be specific for RSV F glycoprotein and bind with high apparent affinity (2 x 10(8) M-1). The other binds with lower affinity and exhibits cross-reactivity with other antigens. CONCLUSION: The strategy described, involving construction of combinatorial IgA libraries on the surface of filamentous phage, should be generally applicable to the investigation of both mucosal and systemic human IgA immune responses, and may become an important tool for evaluation of mucosal vaccine regimes.

Immunoregulation by cytokines.

The study of cytokine biology relevant to the in vivo (patho)physiology of the immune response is complicated by a series of features inherent to the cytokine system. The present survey focuses on the mechanisms governing the function of cytokines that may give rise to methodological and conceptual problems concerning in vivo manipulations of immunologically relevant cytokines. Special emphasis is laid on the complex interrelation between individual cytokines (cascades, synergy, anergy, pleiotropism, and redundancy), as well as on the mechanisms that guarantee a compartmentalization of cytokines, i.e., a chronological, spatial, cell-type differentiation stage, and activation-dependent restriction of their function. The in vivo effects of cytokines can be studied either by augmenting their concentration or reducing their bioavailability. The advantages of local and systemic cytokine injections, usage of transgenes, or expression as gene products encoded by recombinant viruses are discussed and contrasted with different manipulations provoking cytokine deficiencies, namely the application of cytokine antagonists, neutralizing antibodies and receptor derivates, receptor-targeted cytotoxic drugs, and germ line disruption of cytokine genes. Both types of intervention are afflicted with major problems. Whereas providing an excess of cytokines in vivo, especially at the systemic level, constitutes an essentially non-physiological intervention, the induction of cytokine deficiencies will only unravel essential effects, but is incapable of elucidating the many pleiotropic cytokine effects that, by virtue of the redundancy of the system, compensate for each other.

Dissociation of autoaggression and self-superantigen reactivity.

Self-superantigens have been described as products of endogenous retroviruses of the mouse ('minor lymphocyte stimulating loci') that are capable of interacting without prior processing with conserved domains of TCR V beta chains, causing the activation and deletion of most T cells expressing products of determined V beta gene families [1-4]. The fact that superantigens activate a far higher percentage of T cells (1-20%) than conventional, peptidic antigens (< 0.1%) provides the methodological advantage that the degree of clonal deletion may be measured by the analysis of the TCR repertoire using appropriate anti-V beta antibodies. Although much information on the spatio-temporal organization of repertoire-purging has been gathered by virtue of self-superantigens, serious doubts exist as to the possibility that such structures serve as pathogenetically relevant autoantigens. Thus, certain inbred mice spontaneously develop autoimmune diseases, although they bear T-cell repertoires that appear to be purged from self-superantigen-reactive V beta products. In addition, therapeutic interventions targeted to V beta gene products that are not specific for self-superantigens are successful in preventing disease development. The lack of correlation between superantigen-related V beta deletions and autoimmune disease development is substantiated in further models of murine autoimmunity. Based on these observations, we formulate the hypothesis that self-superantigen-reactive T cells are not involved in the development of autoimmune diseases.

Functional double-negative T cells in the periphery express T cell receptor V beta gene products that cause deletion of single-positive T cells.

A proportion of peripheral T cells lack surface expression of the CD4 or CD8 coreceptor molecules and hence are designated as " double negative" (DN). Most DN T lymphocytes express the gamma/delta T cell receptor (TcR), but a minor fraction of them, in both humans and mice, express the alpha/beta TcR. Whereas alpha/beta+ DN T lymphocytes are infrequent (< 1%) in conventional lymphoid organs (spleen, blood, lymph node), they account for two-thirds of the T cells residing in adult bone marrow. Analysis of the TcR V beta repertoire expressed by peripheral DN T cells revealed a high frequency of cells bearing autoreactive TcR that cause deletion of "single-positive" (SP) (CD4+CD8-or CD4-CD8+) T cells. Peripheral DN cells thus represent a cell type that is relatively resistant to clonal deletion. Furthermore, such cells have not been inactivated (anergized) in vivo since they proliferate and secrete interleukins in response to cross-linking by monoclonal antibodies specific for these V beta gene products that are deleted in SPT cells. These results might help to understand the association of peripheral expansion of DN cells and development of autoimmune diseases.

In vivo administration of interleukin-2 turns on anergic self-reactive T cells and leads to autoimmune disease.

One major mechanism of self tolerance involves the deletion of T cell clones in the thymus. In athymic mice, tolerance to self antigens must be generated extrathymically. T cells with self-reactive receptors undergo either peripheral clonal deletion or become unresponsive (i.e. anergic). The unresponsive state of human and mouse T cell clones in vitro can be reversed by the addition of exogenous interleukin (IL)-2, thus transforming anergic T cells to an activated state. Here it is shown that the in vivo delivery of IL-2 to athymic BALB/c nu/nu mice abrogates the anergic state of self-reactive V beta 3+ and V beta 11+ T cells [which are normally deleted in the minor lymphocyte stimulatory (Mls)-1b-, I-E(+)-expressing euthymic counterparts]. Thus, V beta 3+ and V beta 11+ T cells from IL-2-treated nude mice proliferate in response to T cell receptor cross-linking and acquire effector functions as measured by their ability to deliver aid to B cells upon specific stimulation. This activation correlates with the development of autoimmune manifestations (DNA autoantibodies, rheumatoid factors, erythroleukopenia and minimal change nephritis) in these IL-2-treated mice.

Attenuation of autoimmune disease and lymphocyte accumulation in MRL/lpr mice by treatment with anti-V beta 8 antibodies.

MRL-MP-lpr/lpr mice are afflicted by a severe systemic autoimmune disease that is aggravated by the lpr mutation resulting in the accumulation of phenotypically abnormal lpr cells (CD3+CD4-CD8-) in all lymphoid issues including hyperplastic lymph nodes. Given that products of the T cell receptor V beta 8 gene family are overrepresented among lpr cells, different schedules aimed at selectively decreasing the frequency of lpr cells were designed. First, continuous administration of the monoclonal antibody F23.1 (specific for V beta 8 products) resulted in a significant depletion of V beta 8+ cells and prevented the manifestation of lymph accumulation at the same time as it reduced the serological, clinical, and histopathological signs of autoimmune disease. Along the same line, administration of either F23.1 or two different anti-F23.1 anti-idiotypic antibodies to MRL/Mp-lpr/lpr mothers elicited, in the offspring, the production of antibodies sharing a recurrent idiotype with F23.1 and resulted in long-term amelioration of autoimmunity and lymphadenopathy. Thus, a strategy aimed at specifically reducing the frequency of a subset of lpr cells proved successful in mitigating the autoimmune process.

lpr T cells vaccinate against lupus in MRL/lpr mice.

MRL/MP-lpr/lpr mice are homozygous for the lpr mutation that results in the accumulation of phenotypically abnormal cells (CD3+CD4+CD8-) in all lymphoid issues. Although no major abnormalities in the T cell receptor repertoire expressed by such lpr cells have been reported, the lpr mutation is a major disease-accelerating factor. Finally, intravenous administration of irradiated lpr cells recovered from the hyperplastic lymph nodes of adult diseased animals to young MRL/Mp-lpr/lpr mice resulted in a highly significant amelioration of disease parameters. This "T cell vaccination" approach resulted in a selective depletion of cells expressing products of the V beta 8.2 subfamily among lymph node T cells, in addition to eliciting a surge in peripheral T cells capable of conferring disease protection in adoptive transfer experiments. Thus, a strategy aimed at specifically reducing the frequency of lpr cells proved successful in mitigating the autoimmune process. These findings add to the involvement of lpr cells in the autoimmune process and constitute the first report that T cell vaccination may be beneficial to a spontaneously occurring autoimmune disease.

Expansion and clonal deletion of peripheral T cells induced by bacterial superantigen is independent of the interleukin-2 pathway.

Injection of the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) into mice provokes a rapid expansion and subsequent contraction of the pool of SEB-reactive T cells bearing T cell receptor (TcR) V beta 8 gene products. Given that interleukin 2 (IL-2) stimulates proliferation, abolishes anergy, and counteracts apoptotic cell death in T cells in vitro, we tested whether the IL-2 synthesis inhibitor cyclosporin A (CsA) or a vaccinia virus recombinant releasing high amounts of human IL-2 modulate SEB responses in vivo. Surprisingly, neither IL-2 nor CsA were able to change the in vivo kinetics and magnitude of SEB-induced expansion, unresponsiveness to SEB, and peripheral clonal deletion of T cells expressing products of the SEB-reactive TcR V beta 8 gene family. In accord with these in vivo observations, IL-2 is incapable of reversing "anergy" and apoptotic cell death of V beta 8+ SEB-reactive T cells isolated from SEB-primed mice in vitro. Accordingly, upon SEB injection V beta 8+ T cells expand rapidly, without expressing IL-2 receptor (IL-2R)alpha chains in vivo, although SEB induces IL-2R alpha in vitro. Altogether, these results indicate that the IL-2/IL-2R-mediated pathway is not involved in T cell repertoire modulation by bacterial superantigens. Moreover, the data suggest that unresponsiveness of V beta 8+ T cells from SEB-primed mice is not a reversible process, but involves an unreversible commitment to programmed cell death. Absence or presence of IL-2 responsiveness could be a hallmark to distinguish truly reversible anergy and peripheral clonal deletion.

Interleukin 2 abrogates the nonresponsive state of T cells expressing a forbidden T cell receptor repertoire and induces autoimmune disease in neonatally thymectomized mice.

Under physiological conditions, the vast majority of T cells differentiate in the thymus, an organ that provides an optimal microenvironment for T cell maturation and shapes the T cell repertoire via positive and negative selection processes. In the present report, we demonstrate that neonatal thymectomy of CBA/H mice results in a diminution of T cells in peripheral lymphoid organs (spleen, lymph nodes), but is followed by a marked transient (12 wk) increase in Thy-1+ CD3+ cells in the peritoneal cavity. These cells exhibit predominantly a double-negative (CD4-CD8-) phenotype among which products of the T cell receptor (TCR) V beta 11 gene family (i.e., an I-E-reactive TCR normally deleted in I-E-bearing CBA/H mice) are selectively overexpressed. This observation suggests that, under athymic conditions, T cell differentiation and/or accumulation may occur in the peritoneal cavity. Intraperitoneal inoculation of an interleukin 2 (IL-2) vaccinia virus construct that releases high titers of human IL-2 in vivo induces conversion of these double-negative T cells to either CD4+ CD8- or CD4- CD8+ single positives, and allows in vitro stimulation of TCR V beta 11-bearing cells with a clonotypic anti-V beta antibody. Since IL-2 induces autoimmune manifestations (DNA autoantibodies, rheumatoid factors, and interstitial nephritis) in thymectomized CBA/H mice, but not in sham-treated littermates, this lymphokine is likely to enhance the autoaggressive function of T cells that bear forbidden, potentially autoreactive TCR gene products and that are normally deleted in the thymus.