The stress kinase mitogen-activated protein kinase kinase (MKK)7 is a negative regulator of antigen receptor and growth factor receptor-induced proliferation in hematopoietic cells.

The dual specificity kinases mitogen-activated protein kinase (MAPK) kinase (MKK)7 and MKK4 are the only molecules known to directly activate the stress kinases stress-activated protein kinases (SAPKs)/c-Jun N-terminal kinases (JNKs) in response to environmental or mitogenic stimuli. To examine the physiological role of MKK7 in hematopoietic cells, we used a gene targeting strategy to mutate MKK7 in murine T and B cells and non-lymphoid mast cells. Loss of MKK7 in thymocytes and mature B cells results in hyperproliferation in response to growth factor and antigen receptor stimulation and increased thymic cellularity. Mutation of mkk7 in mast cells resulted in hyperproliferation in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF). SAPK/JNK activation was completely abolished in the absence of MKK7, even though expression of MKK4 was strongly upregulated in mkk7(-/-) mast cell lines, and phosphorylation of MKK4 occurred normally in response to multiple stress stimuli. Loss of MKK7 did not affect activation of extracellular signal-regulated kinase (ERK)1/2 or p38 MAPK. mkk7(-/-) mast cells display reduced expression of JunB and the cell cycle inhibitor p16INK4a and upregulation of cyclinD1. Reexpression of p16INK4a in mkk7(-/-) mast cells abrogates the hyperproliferative response. Apoptotic responses to a variety of stimuli were not affected. Thus, MKK7 is an essential and specific regulator of stress-induced SAPK/JNK activation in mast cells and MKK7 negatively regulates growth factor and antigen receptor-driven proliferation in hematopoietic cells. These results indicate that the MKK7-regulated stress signaling pathway can function as negative regulator of cell growth in multiple hematopoietic lineages.

CD45: new jobs for an old acquaintance.

Identified as the first and prototypic transmembrane protein tyrosine phosphatase (PTPase), CD45 has been extensively studied for over two decades and is thought to be important for positively regulating antigen-receptor signaling via the dephosphorylation of Src kinases. However, new evidence indicates that CD45 can function as a Janus kinase PTPase that negatively controls cytokine-receptor signaling. A point mutation in CD45, which appears to affect CD45 dimerization, and a genetic polymorphism that affects alternative CD45 splicing are implicated in autoimmunity in mice and multiple sclerosis in humans. CD45 is expressed in multiple isoforms and the modulation of specific CD45 splice variants with antibodies can prevent transplant rejections. In addition, loss of CD45 can affect microglia activation in a mouse model for Alzheimer's disease. Thus, CD45 is moving rapidly back into the spotlight as a drug target and central regulator involved in differentiation of multiple hematopoietic cell lineages, autoimmunity and antiviral immunity.

Function of PI3Kgamma in thymocyte development, T cell activation, and neutrophil migration.

Phosphoinositide 3-kinases (PI3Ks) regulate fundamental cellular responses such as proliferation, apoptosis, cell motility, and adhesion. Viable gene-targeted mice lacking the p110 catalytic subunit of PI3Kgamma were generated. We show that PI3Kgamma controls thymocyte survival and activation of mature T cells but has no role in the development or function of B cells. PI3Kgamma-deficient neutrophils exhibited severe defects in migration and respiratory burst in response to heterotrimeric GTP-binding protein (G protein)-coupled receptor (GPCR) agonists and chemotactic agents. PI3Kgamma links GPCR stimulation to the formation of phosphatidylinositol 3,4,5-triphosphate and the activation of protein kinase B, ribosomal protein S6 kinase, and extracellular signal-regulated kinases 1 and 2. Thus, PI3Kgamma regulates thymocyte development, T cell activation, neutrophil migration, and the oxidative burst.

OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis.

The tumour-necrosis-factor-family molecule osteoprotegerin ligand (OPGL; also known as TRANCE, RANKL and ODF) has been identified as a potential osteoclast differentiation factor and regulator of interactions between T cells and dendritic cells in vitro. Mice with a disrupted opgl gene show severe osteopetrosis and a defect in tooth eruption, and completely lack osteoclasts as a result of an inability of osteoblasts to support osteoclastogenesis. Although dendritic cells appear normal, opgl-deficient mice exhibit defects in early differentiation of T and B lymphocytes. Surprisingly, opgl-deficient mice lack all lymph nodes but have normal splenic structure and Peyer's patches. Thus OPGL is a new regulator of lymph-node organogenesis and lymphocyte development and is an essential osteoclast differentiation factor in vivo.

Thymic epithelial cells synthesize a heparan sulfate with a highly sulfated region.

Epithelial cells are important components of the thymus microenvironment and are involved in thymocyte differentiation. The production and secretion of sulfated glycosaminoglycans by these cells grown in culture were investigated using labeling with radioactive 35S-Na2SO4 and 3H-glucosamine. The major glycosaminoglycans synthesized by these cells are heparan sulfate and hyaluronic acid. The structure of the heparan sulfate was investigated by the pattern of degradation products formed by deaminative cleavage with nitrous acid. The ratio 35S-sulfate/ H-glucosamine is high in the segments of the heparan sulfate released during the deaminative cleavage with nitrous acid but low in the resistant portion of the molecule. Thus, the heparan sulfate synthesized by the thymic epithelial cells contains a highly sulfated region. Digestion with heparitinase reveals that this highly sulfated region is a heparin-like segment of the molecule. The heparan sulfate is rapidly incorporated into the cell surface but its secretion to the extracellular medium requires a longer incubation period. Finally, heparin was used to mimic the possible effect of this heparan sulfate with a highly sulfated region, as ascertained by its ability to modulate thymocyte adhesion to thymic epithelial cells. Since heparin actually enhanced thymocyte adhesion, it is suggested that the heparan sulfate described herein, secreted by the thymic epithelium, may play a role upon intrathymic heterotypic cellular interactions.

Thymic heterotypic cellular complexes in gene-targeted mice with defined blocks in T cell development and adhesion molecule expression.

Thymocytes form unique multicellular complexes with epithelial cells (thymic nurse cells, TNC) and rosettes (ROS) with macrophages, epithelial cells and dendritic cells. To investigate the role of differentiation checkpoints in the formation of the thymic heterotypic complexes in vivo, we used mutant mice which have genetically defined blocks at early and late stages of T cell development. We show that RAG-1-/-, TCRbeta-/- , and p56lck-/- mice lack thymocyte ROS formation with epithelial cells, macrophages, or dendritic cells. TNC formation was not affected by TCRbeta and p56lck gene mutations but partially decreased in RAG-1-/- mice, indicating that TNC are the earliest thymocyte-stromal cell complexes formed in development, whereas ROS only appear after thymocytes have rearranged and expressed a functional TCRbeta chain. Genetic blocks in CD8 lineage commitment (CD8-/- and IFN regulatory factor-1-/- mice) and positive and negative T cell selection (CD45-/-, TCRalpha-/-, and CD30-/- mice) did not affect thymocyte-stromal cell complexes. Surprisingly, CD4-/- mice, but not MHC class II-/- mice, had significantly reduced numbers of TNC and ROS, in particular, a severe defect in ROS formation with thymic dendritic cells. The CD4-/- block in ROS and TNC formation was rescued by the introduction of a human CD4 transgene. Moreover, we show that the adhesion receptors CD44 and LFA-1 cooperate in the formation of the thymic microenvironment. These results provide genetic evidence on the role of defined stages in T cell development and adhesion molecules on thymocyte/stromal cell interactions in vitro.

The inositol polyphosphate 5-phosphatase ship is a crucial negative regulator of B cell antigen receptor signaling.

Ship is an Src homology 2 domain containing inositol polyphosphate 5-phosphatase which has been implicated as an important signaling molecule in hematopoietic cells. In B cells, Ship becomes associated with Fcgamma receptor IIB (FcgammaRIIB), a low affinity receptor for the Fc portion of immunoglobulin (Ig)G, and is rapidly tyrosine phosphorylated upon B cell antigen receptor (BCR)-FcgammaRIIB coligation. The function of Ship in lymphocytes was investigated in Ship-/- recombination-activating gene (Rag)-/- chimeric mice generated from gene-targeted Ship-/- embryonic stem cells. Ship-/-Rag-/- chimeras showed reduced numbers of B cells and an overall increase in basal serum Ig. Ship-/- splenic B cells displayed prolonged Ca2+ influx, increased proliferation in vitro, and enhanced mitogen-activated protein kinase (MAPK) activation in response to BCR-FcgammaRIIB coligation. These results demonstrate that Ship plays an essential role in FcgammaRIIB-mediated inhibition of BCR signaling, and that Ship is a crucial negative regulator of Ca2+ flux and MAPK activation.

Murine thymic nurse cells and rosettes: analysis of adhesion molecule expression using confocal microscopy and a simplified enrichment method.

Thymic nurse cells (TNC) and T-cell stromal rosettes (ROS) are two in vivo models for stromal cell-thymocyte interactions. We describe a simplified enrichment method for TNC and ROS that overcomes the necessity for large amounts of tissue. The complexes were further analyzed with confocal microscopy, and three subunits of ROS were defined on the basis of their central cell phenotype, i.e., macrophage, dendritic, or epithelial cell rosettes. Because adhesion molecules are proposed to play a crucial role in T-cell development, we investigated CD44, LFA-1, and ICAM-1 expression in such complexes. The epithelial component of TNC expresses CD44 and ICAM-1, whereas intra-TNC thymocytes are LFA-negative. With regard to ROS, all subsets expressed CD44, and macrophage and dendritic cell ROS were also ICAM-1-positive and LFA-1-positive. The current protocol opens the possibility for further in vivo analysis of stromal cell-thymocyte interactions, e.g., for studies of scarce gene mutant mice.

Development of spontaneous autoimmune thyroiditis in Obese strain (OS) chickens.

Chickens of the Obese strain (OS) are known to develop spontaneous autoimmune thyroiditis (SAT) in the first 2-3 weeks post-hatching, but onset and severity of SAT for this period among sublines of OS chickens has not yet been analysed in detail. In the present paper, we described the kinetics of SAT in age-matched OSB13B13C, OSB5B5C, OSB15B15INN and OSB5B5INN chicken sublines. The results revealed no thyroid infiltration in OS fetuses at 20th ED of the analysed sublines. Mononuclear cell infiltration of thyroid was first noted in 2-4-day-old chickens, followed by aggravation of thyroiditis in 4-week-old OSB13B13C and OSB15B15INN chickens and in 5-week-old OSB5B5C birds. Interestingly, two subpopulations of OSB5B5INN chickens were found with different kinetics of SAT development: one with degree of SAT lower than 40%, was designated "low responders" and the other, with similar degrees of SAT as the other three sublines, was characterized as "high responders". Our results allow an age-dependent prediction of SAT development among OS chickens and the rational design of animal experiments, particularly for assessing the relevance of therapeutic interventions.