Interleukin-5 (IL-5) and IL-6 define two molecularly distinct pathways of B-cell differentiation.

Interleukin-5 (IL-5) and IL-6 have both been reported to act as B-cell differentiation factors by stimulating activated B cells to secrete antibody. However, it has not been possible to directly compare the effects of these two lymphokines because of the lack of a suitable B-cell line capable of responding to both. We have identified a clonal, inducible B-cell lymphoma, CH12, that has this property. Both IL-5 and IL-6 can independently stimulate increases in steady-state levels of immunoglobulin and J-chain mRNA and proteins, and they both induce the differentiation of CH12 into high-rate antibody-secreting cells. Nevertheless, there are significant differences in the activities of these two lymphokines. First, while IL-6 acts only as a differentiation factor, IL-5 also augments the proliferation of CH12 cells. Second, the differentiation stimulated by IL-5 but not by IL-6 is partially inhibited by IL-4. Inhibition of IL-5-induced differentiation was not at the level of IL-5 receptor expression, since IL-4 did not inhibit IL-5-induced proliferation. Third, IL-5 but not IL-6 stimulated increased mouse mammary tumor proviral gene expression in CH12 cells. These results demonstrate that while both IL-5 and IL-6 may act as differentiation factors for B cells, they induce differentiation by using at least partially distinct molecular pathways. Our results also establish that B cells characteristic of a single stage of development can independently respond to IL-4, IL-5, and IL-6.     

The novel adaptor protein Swiprosin-1 enhances BCR signals and contributes to BCR-induced apoptosis

B-cell receptor (BCR) signals are essential for B-cell differentiation, homeostasis and negative selection, which are regulated by the strength and quality of BCR signals. Recently, we identified a new adaptor protein, Swiprosin-1, in lipid rafts of B-cell lines that undergo apoptosis after BCR stimulation. During murine B-cell development, Swiprosin-1 exhibited highest expression in immature B cells of the bone marrow, but was also expressed in resting and activated splenic B cells and in non-lymphoid tissue, especially in the brain. Ectopic expression of Swiprosin-1 in the immature murine B-cell line WEHI231 enhanced spontaneous and BCR-induced apoptosis. In contrast, short hairpin RNA (shRNA)-mediated downregulation of Swiprosin-1 impaired specifically spontaneous and BCR-elicited apoptosis, but not BCR-induced G1 cell cycle arrest and upregulation of the cell cycle inhibitor p27(Kip1). In accordance, Swiprosin-1 abundance regulated net cell growth of WEHI231 cell populations through reciprocal regulation of Bcl-xL, but not Bim, thereby controlling spontaneous apoptosis. Swiprosin-1-enhanced apoptosis was blocked through nuclear factor kappaB-activating stimuli, namely B-cell-activating factor of the TNF family, anti-CD40 and lipopolysaccharide (LPS). This correlated with enhanced BCR-induced IkappaB-alpha phosphorylation and degradation in cells expressing a Swiprosin-1-specific shRNA. Finally, ectopic Swiprosin-1 expression enhanced BCR-induced cell death in primary, LPS-stimulated splenic B cells. Hence, Swiprosin-1 may regulate lifespan and BCR signaling thresholds in immature B cells.     

Glucocorticoid-resistant lymphoma cell variants that contain functional glucocorticoid receptors.

A mouse T-lymphosarcoma cell line stably infected with mouse mammary tumor virus (MMTV) was used as the parent line for a genetic analysis of two glucocorticoid hormone responses, hormone-induced cytolysis and stimulation of viral gene expression. Variants were selected for survival and elevated expression of MMTV proteins in the presence of the steroid. The MMTV marker provided a sensitive test for glucocorticoid receptor (GR) function in the hormone-resistant variants. This strategy resulted in the isolation of two novel types of hormone-resistant variants. One type of variant with only about 25% of the level of GR found in the parent line was resistant to the cytolytic effects of glucocorticoid but produced increased levels of MMTV gene products in response to the hormone. This variant phenotype demonstrated that the MMTV response requires fewer GR than the cytolytic response. Another variant, which required approximately 100-fold higher concentrations of hormone than the wild-type cells for both responses, apparently contained GR with altered hormone-binding properties.     

Role of dendritic cells in the immune response induced by mouse mammary tumor virus superantigen.

After mouse mammary tumor virus (MMTV) infection, B lymphocytes present a superantigen (Sag) and receive help from the unlimited number of CD4(+) T cells expressing Sag-specific T-cell receptor Vbeta elements. The infected B cells divide and differentiate, similarly to what occurs in classical B-cell responses. The amplification of Sag-reactive T cells can be considered a primary immune response. Since B cells are usually not efficient in the activation of naive T cells, we addressed the question of whether professional antigen-presenting cells such as dendritic cells (DCs) are responsible for T-cell priming. We show here, using MMTV(SIM), a viral isolate which requires major histocompatibility complex class II I-E expression to induce a strong Sag response in vivo, that transgenic mice expressing I-E exclusively on DCs (I-EalphaDC tg) reveal a strong Sag response. This Sag response was dependent on the presence of B cells, as indicated by the absence of stimulation in I-EalphaDC tg mice lacking B cells (I-EalphaDC tg muMT(-/-)), even if these B cells lack I-E expression. Furthermore, the involvement of either residual transgene expression by B cells or transfer of I-E from DCs to B cells was excluded by the use of mixed bone marrow chimeras. Our results indicate that after priming by DCs in the context of I-E, the MMTV(SIM) Sag can be recognized on the surface of B cells in the context of I-A. The most likely physiological relevance of the lowering of the antigen threshold required for T-cell/B-cell collaboration after DC priming is to allow B cells with a low affinity for antigen to receive T-cell help in a primary immune response.     

Microarray Analyses of Glucocorticoid and Vitamin D3 Target Genes in Differentiating Cultured Human Podocytes

Glomerular podocytes are highly differentiated epithelial cells that are key components of the kidney filtration units. Podocyte damage or loss is the hallmark of nephritic diseases characterized by severe proteinuria. Recent studies implicate that hormones including glucocorticoids (ligand for glucocorticoid receptor) and vitamin D3 (ligand for vitamin D receptor) protect or promote repair of podocytes from injury. In order to elucidate the mechanisms underlying hormone-mediated podocyte-protecting activity from injury, we carried out microarray gene expression studies to identify the target genes and corresponding pathways in response to these hormones during podocyte differentiation. We used immortalized human cultured podocytes (HPCs) as a model system and carried out in vitro differentiation assays followed by dexamethasone (Dex) or vitamin D3 (VD3) treatment. Upon the induction of differentiation, multiple functional categories including cell cycle, organelle dynamics, mitochondrion, apoptosis and cytoskeleton organization were among the most significantly affected. Interestingly, while Dex and VD3 are capable of protecting podocytes from injury, they only share limited target genes and affected pathways. Compared to VD3 treatment, Dex had a broader and greater impact on gene expression profiles. In-depth analyses of Dex altered genes indicate that Dex crosstalks with a broad spectrum of signaling pathways, of which inflammatory responses, cell migration, angiogenesis, NF-κB and TGFβ pathways are predominantly altered. Together, our study provides new information and identifies several new avenues for future investigation of hormone signaling in podocytes.     

Differential glycosylation of two glycoproteins synthesized by murine B cells in response to IL-4 plus IL-5

We sought to determine whether selected cytokines, known to stimulate profoundly B-cell activation and differentiation, also have as yet unrecognized effects upon the glycosylation of secreted Ig and/or membrane-associated proteins. The glycosylation of both secreted IgM and membrane-bound MHC Class-I synthesized by CH12LX cells was detected by enzyme-lectin conjugates in immunoabsorption assays. Stimulation of B cells with IL-4 plus IL-5 significantly decreases the terminal glycosylation of secreted IgM, whereas LPS has a minor effect, despite the fact that both stimuli are equipotent for IgM secretion. Neither LPS nor IL-4 plus IL-5 affect MHC Class-I expression. However, IL-4 plus IL-5 substantially increases the terminal glycosylation of MHC Class-I produced from both mIgM(+)and mIgA(+)CH12LX cells. LPS has no or a modest effect on the terminal glycosylation of MHC Class-I produced from CH12LX cells. These results suggest that Th(2)-derived cytokines differentially influence the glycosylation of secreted and membrane-associated glycoproteins of B cells. In turn, this might elucidate the basis of aberrant glycosylation reported in conditions such as IgA nephropathy, cancer and rheumatoid arthritis.