Individual and overlapping roles of BH3-only proteins Bim and Bad in apoptosis of lymphocytes and platelets and in suppression of thymic lymphoma development.

BH3-only proteins, such as Bim and Bad, contribute to tissue homeostasis by initiating apoptosis in a cell type- and stimulus-specific manner. Loss of Bim provokes lymphocyte accumulation in vivo and renders lymphocytes more resistant to diverse apoptotic stimuli and Bad has been implicated in the apoptosis of haematopoietic cells upon cytokine deprivation. To investigate whether their biological roles in apoptosis overlap, we generated mice lacking both Bim and Bad and compared their haematopoietic phenotype with that of the single-knockout and wild-type (wt) animals. Unexpectedly, bad(-/-) mice had excess platelets due to prolonged platelet life-span. The bim(-/-)bad(-/-) mice were anatomically normal and fertile. Their haematopoietic phenotype resembled that of bim(-/-) mice but lymphocytes were slightly more elevated in their lymph nodes. Although resting B and T lymphocytes from bim(-/-)bad(-/-) and bim(-/-) animals displayed similar resistance to diverse apoptotic stimuli, mitogen activated bim(-/-)bad(-/-) B cells were more refractory to cytokine deprivation. Moreover, combined loss of Bim and Bad-enhanced survival of thymocytes after DNA damage and accelerated development of γ-irradiation-induced thymic lymphoma. Unexpectedly, their cooperation in the thymus depended upon thymocyte-stromal interaction. Collectively, these results show that Bim and Bad can cooperate in the apoptosis of thymocytes and activated B lymphocytes and in the suppression of thymic lymphoma development.

Defective gp130-mediated signal transducer and activator of transcription (STAT) signaling results in degenerative joint disease, gastrointestinal ulceration, and failure of uterine implantation.

The receptor subunit gp130 transduces multiple cell type-specific activities of the leukemia inhibitory factor (LIF)/interleukin (IL)-6 family of cytokines through the signal transducer and activator of transcription (STAT) and src homology 2 domain-bearing protein tyrosine phosphatase (SHP)-2/ras/Erk pathways. To define STAT-dependent physiological responses, we generated mice with a COOH-terminal gp130(DeltaSTAT) "knock-in" mutation which deleted all STAT-binding sites. gp130(DeltaSTAT) mice phenocopyed mice deficient for IL-6 (impaired humoral and mucosal immune and hepatic acute phase responses) and LIF (failure of blastocyst implantation). However, unlike mice with null mutations in any of the components in the gp130 signaling pathway, gp130(DeltaSTAT) mice also displayed gastrointestinal ulceration and a severe joint disease with features of chronic synovitis, cartilaginous metaplasia, and degradation of the articular cartilage. Mitogenic hyperresponsiveness of synovial cells to the LIF/IL-6 family of cyto-kines was caused by sustained gp130-mediated SHP-2/ras/Erk activation due to impaired STAT-mediated induction of suppressor of cytokine signaling (SOCS) proteins which normally limits gp130 signaling. Therefore, the joint pathology in gp130(DeltaSTAT) mice is likely to arise from the disturbance of the otherwise balanced activation of the SHP-2/ras/Erk and STAT signaling cascades emanating from gp130.

Single cell sorting and cloning.

Cell sorters now allow the selection of cells and other bodies according to a range of quite diverse criteria. The additional refinement that allows the sorting of individual cells based on these criteria has seen application in many fields of research. Single cells may be sorted for microscopy, for culture and for genetic analysis by way of single cell PCR (polymerase chain reaction). In practical terms, in the setting up of an instrument for single cell sorting, there are additional requirements to ensure that each detected event is indeed a single cell or body, that this cell can be reliably sorted via saline droplet, separate from its fellow travelers, that the aiming of the droplet deflection is sufficiently precise to find the target vessel and that the cell will be undamaged on arrival. Among the diverse reported applications of the technique, two fields which have benefited greatly are lymphocyte development and haemopoiesis. In the former case, the analysis of gene rearrangements in lymphocytes, both in the pre- and post-antigenic phases of development, has been enabled by the combined technologies of single cell sorting and PCR. It is argued that such experiments could not have been done without that partnership. In a similar way, the single cell sorting technique has been found to be the perfect way to demonstrate precursor/progeny relationships between haemopoietic cells and, further, to demonstrate rigorously the effects of particular cytokines on the haemopoietic system.

Dissecting affinity maturation: a model explaining selection of antibody-forming cells and memory B cells in the germinal centre.

Until recently, the relationship between apoptosis, selection in the germinal centre (GC) and production of high-affinity antibody-forming cells (AFCs) and memory B cells has been unclear. Here, Tarlinton and Smith present a model that accounts for the switch in GC production from high-affinity AFCs to memory B cells, and explain how Bcl-2, an inhibitor of apoptosis, can influence memory cells but not bone marrow AFCs.

Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity.

Apoptosis can be triggered by members of the Bcl-2 protein family, such as Bim, that share only the BH3 domain with this family. Gene targeting in mice revealed important physiological roles for Bim. Lymphoid and myeloid cells accumulated, T cell development was perturbed, and most older mice accumulated plasma cells and succumbed to autoimmune kidney disease. Lymphocytes were refractory to apoptotic stimuli such as cytokine deprivation, calcium ion flux, and microtubule perturbation but not to others. Thus, Bim is required for hematopoietic homeostasis and as a barrier to autoimmunity. Moreover, particular death stimuli appear to activate apoptosis through distinct BH3-only proteins.

Kinetics of establishing the memory B cell population as revealed by CD38 expression.

In this report, we detail changes in the expression of CD38 on murine B cells during the course of a T cell-dependent immune response. CD38 is expressed on all naive B cells but is down-regulated on isotype-switched B cells from both the germinal centers (GCs) and the foci of Ab-forming cells which arise during the first weeks of the response. The down-regulation on GC B cells, however, is reversible since Ag-specific IgG1 B cells with high levels of CD38 are apparent by 2 wk postimmunization. These cells have characteristics that resemble recirculating memory B cells, in that they are small and bind low levels of peanut agglutinin. Such characteristics indicate that the restoration of CD38 levels is coincidental with the transition from GC to memory B cell. Using this observation, we plotted the development of the memory population and the demise of the GC reaction as a function of time after immunization. Our results indicate that the GC reaction ceases gradually over many weeks rather than suddenly, which corresponds with the formation of the memory B cell population. Furthermore, by segregating memory B cells and GC B cells, it was possible to assess the in vitro survival characteristics of each compared with naive B cells. These experiments demonstrated that memory B cell survival in vitro was comparable with naive B cell survival but less than the survival seen for bcl-2-transgenic B cells, whereas GC B cell survival, as expected, was very poor. Hence, by resolving murine Ag-specific memory B cells and GC B cells, we have been able to quantify the development of the memory B cell population.

Inhibition of the B cell by CD22: a requirement for Lyn.

Mice in which the Lyn, Cd22, or Shp-1 gene has been disrupted have hyperactive B cells and autoantibodies. We find that in the absence of Lyn, the ability of CD22 to become tyrosine phosphorylated after ligation of mIg, to recruit SHP-1, and to suppress mIg-induced elevation of intracellular [Ca2+] is lost. Therefore, Lyn is required for the SHP-1-mediated B cell suppressive function of CD22, accounting for similarities in the phenotypes of these mice.

Continued differentiation during B lymphopoiesis requires signals in addition to cell survival.

During B lymphopoiesis, cells undergo successive rounds of division and growth arrest coupled to intermittent selection on the basis of Ig expression. It is unresolved whether differentiation requires specific signaling or is merely the consequence of sustained cell survival. Transgenic expression of the cell death antagonist, Bcl-2, promoted accumulation of B lymphoid cells in mice deficient in antigen receptor rearrangement (scid or rag-1-/-) and in mice lacking the IgM transmembrane domain (microMT). Continued differentiation occurred, however, only in the bcl-2/scid and bcl-2/microMT mice. The appearance of B lineage cells expressing CD21, CD22 and CD23 was associated with DHJH rearrangements which encode a truncated C mu-containing protein called D mu in bcl-2/scid mice and with expression of Ig heavy chain classes other than IgM in the bcl-2/ microMT mice. In neither case, however, were proliferating cells observed in the more mature B lineage compartments in the bone marrow. Thus, continued B cell development requires signaling via Ig heavy chain-containing receptors and is not simply a consequence of blocking apoptosis.

Expression of a bcl-2 transgene reduces proliferation and slows turnover of developing B lymphocytes in vivo.

B lymphocyte differentiation proceeds through a series of alternating stages of proliferative expansion interspersed with noncycling stationary phases during which cells undergo either positive selection or apoptotic cell death. The molecular control of cell cycle progression and that of apoptosis appear to be interconnected. Overexpression of Bcl-2 in lymphocytes or fibroblasts antagonizes apoptosis and delays their transition from the quiescent state into the cell cycle. We have undertaken a systematic analysis of the impact of bcl-2 transgene expression on cell cycle distribution and turnover rate of developing B lymphocytes in normal mice and in mutant animals in which B cell differentiation is arrested at the pro-B/pre-BI or the pre-BII stage. These experiments revealed that overexpression of Bcl-2 reduces proliferation and slows turnover of B cells at all stages of development. This demonstrates that Bcl-2 can retard transition of B cells between the quiescent and the cycling state regardless of the mitogenic stimulus and the differentiation stage. The implications of these results for the normal control of B lymphopoiesis and for lymphomagenesis are discussed.