Ramón Merino

Developmental regulation of the Bcl-2 protein and susceptibility to cell death in B lymphocytes.

Cell death is a prominent feature of B cell development. For example, a large population of B cells dies at the pre‐B cell stage presumably due to the failure to express a functional immunoglobulin receptor. In addition, developing B cells expressing antigen receptors for self are selectively eliminated at the immature B cell stage. The molecular signals that control B cell survival are largely unknown. The product of the bcl‐2 proto‐oncogene may be involved as its overexpression inhibits apoptotic cell death in a variety of biological systems. However, the physiological role of the endogenous Bcl‐2 protein during B cell development is undetermined. Here we show a striking developmental regulation of the Bcl‐2 protein in B lymphocytes. Bcl‐2 is highly expressed in CD43+ B cell precursors (pro‐B cells) and mature B cells but downregulated at the pre‐B and immature B cell stages of development. We found that Bcl‐2 expressed by B cells is a long‐lived protein with a half‐life of approximately 10 h. Importantly, susceptibility to apoptosis mediated by the glucocorticoid hormone dexamethasone is stage‐dependent in developing B cells and correlates with the levels of Bcl‐2 protein. Furthermore, expression of a bcl‐2 transgene rescued pre‐B and immature B cells from dexamethasone‐induced cell death, indicating that Bcl‐2 can inhibit the apoptotic cell death of progenitors and early B cells. Taken together, these findings argue that Bcl‐2 is a physiological signal controlling cell death during B cell development.

Imbalance towards Th1 predominance is associated with acceleration of lupus-like autoimmune syndrome in MRL mice.

To investigate the respective roles of Th1 and Th2 cells in the pathogenesis of lupus-like autoimmune disease, we have analyzed the spontaneous and antigen-induced productions of IgG1 vs IgG2a and IgG3 subclasses in relation to the mRNA expression of INF-gamma (Th1 cytokine promoting IgG2a and IgG3 production), IL-4 (Th2 cytokine promoting IgG1 production), and IL-10 (Th2 cytokine) in CD4+ T cells from lupus-prone MRL mice. For this purpose, two paired sets of MRL mice were chosen for the comparison of these parameters: (a) MRL-lpr/lpr (lpr for lymphoproliferation) and its recently described substrain with a prolonged survival, termed MRL-lpr/lpr.ll (ll for long lived) and (b) MRL male mice bearing the Yaa (Y-linked autoimmune acceleration) gene (MRL.Yaa) with an accelerated disease and their male counterparts lacking the Yaa gene. We demonstrate herein that the accelerated development of lupus-like autoimmune disease in MRL-lpr/lpr and MRL.Yaa mice, as compared with MRL-lpr/lpr.ll and MRL-+/+ mice, respectively, was correlated with an enhanced expression of IFN-gamma vs IL-4 and IL-10 mRNA in CD4+ T cells, which paralleled with an increase of spontaneous and foreign T cell-dependent antigen-induced productions of IgG2a and IgG3 vs IgG1 antibodies. These data suggest that an imbalance towards Th1 predominance may play a significant role in the acceleration of lupus-like autoimmune disease in MRL mice.

Calcineurin inhibitors, but not rapamycin, reduce percentages of CD4+CD25+FOXP3+ regulatory T cells in renal transplant recipients.

Background. Immunosuppression in renal transplantation, although manageable in the short-term, is a major hurdle for long-term graft survival. Recently, increased frequencies of CD4+CD25high regulatory T cells (Tregs) have been described as an additional mechanism that induces alloimmune tolerance. Methods. We assessed 64 renal transplant recipients with stable renal function for at least one year. Patients were divided into two groups according to the immunosuppression they were receiving at the moment of the study: one consisted of patients receiving rapamycin (Rapa) but not calcineurin inhibitors (CNI), and the other group received CNI but not Rapa. The Rapa group was further divided into three subgroups according to their previous experience with CNI: CNI-free, CNI withdrawal, and CNI conversion. Frequencies of blood Tregs were studied by flow cytometry after staining with monoclonal antibodies specific for different markers of Tregs. Results. Frequencies of CD4+ T cells with regulatory phenotype and function were significantly decreased in peripheral blood of renal transplant patients receiving CNI compared with those receiving Rapa. This effect was independent of an early exposure to CNI because the CNI-free patients in the Rapa group showed similar frequencies of Tregs to the CNI withdrawal and CNI conversion groups. Conclusions. CNI, but not Rapa, induce a decrease of circulating Tregs in stable renal transplant recipients. Thus, Rapa might be further explored in strategies using preservation of Tregs for transplant tolerance. Furthermore, quantification of blood Tregs may be a suitable tool to identify renal transplant recipients who may be candidates for reduced immunosuppression.

Analysis of the molecular cascade responsible for mesodermal limb chondrogenesis: sox genes and BMP signaling

Here, we have studied how Sox genes and BMP signaling are functionally coupled during limb chondrogenesis. Using the experimental model of TGFbeta1-induced interdigital digits, we dissect the sequence of morphological and molecular events during in vivo chondrogenesis. Our results show that Sox8 and Sox9 are the most precocious markers of limb cartilage, and their induction is independent and precedes the activation of BMP signaling. Sox10 appears also to cooperate with Sox9 and Sox8 in the establishment of the digit cartilages. In addition, we show that experimental induction of Sox gene expression in the interdigital mesoderm is accompanied by loss of the apoptotic response to exogenous BMPs. L-Sox5 and Sox6 are respectively induced coincident and after the expression of Bmpr1b in the prechondrogenic aggregate, and their activation correlates with the induction of Type II Collagen and Aggrecan genes in the differentiating cartilages. The expression of Bmpr1b precedes the appearance of morphological changes in the prechondrogenic aggregate and establishes a landmark from which the maintenance of the expression of all Sox genes and the progress of cartilage differentiation becomes dependent on BMPs. Moreover, we show that Ventroptin precedes Noggin in the modulation of BMP activity in the developing cartilages. In summary, our findings suggest that Sox8, Sox9, and Sox10 have a cooperative function conferring chondrogenic competence to limb mesoderm in response to BMP signals. In turn, BMPs in concert with Sox9, Sox6, and L-Sox5 would be responsible for the execution and maintenance of the cartilage differentiation program.

Bcl-2 and Bcl-x: regulatory switches for lymphoid death and survival

The survival and death of lymphoid cells is under the control of a genetic program. Cell death is activated at different stages of development and serves to remove unnecessary and autoreactive lymphocytes, as well as to limit the immune response. The survival of cells is regulated by a set of genes that act as repressors of the cell death mechanism. Of these, bcl-2 and bcl-x exhibit a striking pattern of regulation during lymphoid maturation and can inhibit several forms of apoptotic cell death. Here, Gabriel Núñez and colleagues review recent developments in the field, particularly focusing on the role of the Bcl-2 and Bcl-x proteins in regulating lymphoid death and survival.

Retinoic acid regulates programmed cell death through BMP signalling

rogrammed cell death by apoptosis is one of the major driving forces that shape and pattern the organs and tissues of a developing embryo. One of the best model systems for the study of apoptosis is the interdigital cell death (INZ) that occurs during the outgrowth of the vertebrate limb. However, although much has been learned about the cells involved in this process, the molecular mechanisms underlying INZ remain unclear. Retinoic acid (RA) and bone morphogenetic proteins (BMPs) are two of the signalling components known to be involved in INZ. However, the exact relationships between these two factors in controlling cell death remain unknown. Here we show that RA can control INZ by promoting BMP gene expression and simultaneously repressing the chondrogenic potential of BMPs. We found that RA-soaked beads implanted in the interdigital regions of the chick limb bud promoted apoptosis before the onset of physiological INZ (assessed by neutral red vital staining and TUNEL assay; Fig. 1a–c). Furthermore, interdigital web regression was accelerated (Fig. 1d,e). These effects were preceded by upregulation of different members of the BMP gene family (bmp-7, Fig. 1f,g and bmp-4, not shown). In accordance with these findings, the P

The Yaa Gene Model of Systemic Lupus Erythematosus

Discrimination between self and foreign structures operates through an active process that involves several different mechanisms including clonal deletion, clonal anergy and suppression. The failure of these regulatory mechanisms leads to the persistence and activation of potentially self-reactive cells and the development of autoimmune disorders. Such a situation occurs in the case of systemic lupus erythematosus (SLE). a severe chronic autoimmune disease in which antibodies are produced against self components and the resulting immune complexes are involved in the generation of various tissue lesions. Notably, glomerulonephritis, i.e. lupus nephritis, is the major cause of death in patients with SLE. Although the etiology and pathogenesis of SLE are still poorly understood, it is becoming clear that many genetic factors apparently play essential roles in SLE. The discovery of several murine strains that spontaneously develop an autoimmune syndrome resembling human SLE (Theofilopoulus & Dixon 1985) has allowed considerable progress in this field. In particular, these strains have been powerful tools to study the mechanisms by which genetic factors participate in the pathogenesis of SLE. Early genetic studies on New Zealand mice have demonstrated that many individual autoimmune traits segregate independently of each other (Shirai 1982, Izui et al. 1981, Raveche et al. 1981, Bocchieri et al. 1982). This suggests that there is no common genetic defect which causes a predisposition to overall autoimmune responses in the New Zealand strain. However, the clearest examples of the influence of single genes on the development and progression of murine SLE is the efTect of the lpr (lymphoproliferation) and gld (generalized lymphoproliferative disease) genes (Murphy & Roths 1978, Roths et al. 1984). Both mutations not only accelerate the progression of autoimmune

Bone Morphogenetic Proteins Regulate Interdigital Cell Death in the Avian Embryo

The embryonic limb bud provides an excellent model for analyzing the mechanisms that regulate programmed cell death during development. At the time of digit formation in the developing autopod, the undifferentiated distal mesodermal cells may undergo or chondrogenic differentiation or apoptosis depending whether they are incorporated into the future digital rays or into the interdigital spaces. Both chondrogenesis or apoptosis are induced by local BMPS. However, whereas the chondrogenic‐promoting activity of BMPs appears to be regulated through the BMPR‐ 1b receptor, the mechanism by which the BMPs execute the death program remains unknown. The BMP proapoptotic activity requires the expression of members of the msx family of closely related homeobox‐containing genes and is finally mediated by caspase activation, but the nature of the caspase(s) directly responsible for the cell death is also unknown. Finally, other growth factors present in the developing autopod at the stages of digit formation such as members of the FGF and TGFβ families modulate the ability of BMPs to induce cell death or chondrogenesis.

Inhibition of B cell death causes the development of an IgA nephropathy in (New Zealand white x C57BL/6)F(1)-bcl-2 transgenic mice.

Little is known about the pathogenic mechanisms of IgA nephropathy, despite being the most prevalent form of glomerulonephritis in humans. We report in this study that in (New Zealand White (NZW) × C57BL/6)F1 mice predisposed to autoimmune diseases, the expression of a human bcl-2 (hbcl-2) transgene in B cells promotes a CD4-dependent lupus-like syndrome characterized by IgG and IgA hypergammaglobulinemia, autoantibody production, and the development of a fatal glomerulonephritis. Histopathological analysis of glomerular lesions reveals that the glomerulonephritis observed in these animals resembles that of human IgA nephropathy. The overexpression of Bcl-2 in B cells selectively enhances systemic IgA immune responses to T-dependent Ags. Significantly, serum IgA purified from (NZW × C57BL/6)F1-hbcl-2 transgenic mice, but not from nontransgenic littermates, shows reduced levels of galactosylation and sialylation and an increased ability to deposit in the glomeruli, as observed in human patients with IgA nephropathy. Our results indicate that defects in the regulation of B lymphocyte survival associated with aberrant IgA glycosylation may be critically involved in the pathogenesis of IgA nephropathy, and that (NZW × C57BL/6)F1-hbcl-2 Tg mice provide a new experimental model for this form of glomerulonephritis.

The Yaa gene abrogates the major histocompatibility complex association of murine lupus in (NZB x BXSB)F1 hybrid mice.

To investigate the specific contribution of select MHC class II genes on the development of murine lupus, H-2 congenic (NZB x BXSB)F1 hybrid mice bearing either H-2b/b, H-2d/b, or H-2d/d haplotypes were generated. We compared the clinical development (autoantibody production and glomerulonephritis) of systemic lupus erythematosus (SLE) in these three F1 hybrids in the presence or absence of the mutant gene, Yaa (Y chromosome-linked autoimmune acceleration), which normally accelerates the progression of murine SLE. (NZB x BXSB)F1 hybrid female mice bearing either the H-2b/b or H-2d/b haplotype developed a rapid course of severe SLE, while the appearance of disease was markedly delayed in H-2d/d hybrid females. However, in the presence of the Yaa gene, H-2d/d F1 males developed SLE as severe as H-2b/b and H-2d/b F1 males. These data indicate that (a) the conventional H-2b is a haplotype leading to susceptibility for murine SLE, while H-2d is a relatively resistant haplotype; (b) the H-2b haplotype exhibits a dominant effect on autoimmune responses, similar to the classical MHC-linked Ir gene effect; and (c) most strikingly, the Yaa gene totally abrogates the MHC effect on murine lupus in (NZB x BXSB)F1 hybrid mice.