SummaryMice homozygous for the gene lpr develop marked lymphadenopathy and a spectrum of autoantibodies closely resembling that of human systemic lupus erythematosus. The unusual T cell phenotype of the expanded lymphocyte population and the T-dependence of several antibodies in this strain have suggested that primary T cell abnormalities underlie the autoimmune syndrome. Using double chimeras, we now show that expression of the lpr gene in B cells is absolutely necessary for autoantibody production. Combinations of antiThy 1 .2 + C' treated bone marrow from congenic strains of C57BL/6 mice, differing only at the immunoglobulin heavy chain (Igh) and lpr loci, were transferred into lethally irradiated B6/lpr mice . Double chimerism was documented by allotype-specific surface IgD and IgM immunofluorescence assay of peripheral blood and by allotype-specific enzyme-linked immunosorbent assay for total IgM in serum. Despite the presence of both +/+ and lpr B cells, IgM and IgG2a anti-chromatin as well as IgM anti-IgG were entirely the products of lpr B cells. Total serum IgG2a and IgG1 were also dominated by the 1pr phenotype but not to the same extent. A similar experiment using B6/lpr-Igh' recipients confirmed these findings . Additional experiments in which B6/lpr recipients were infused with ratios of donor bone marrow favoring B6 .C20 +/+ over B6/lpr showed that even though +/+ B cells were overrepresented, autoantibodies were only of the lpr allotype . In addition, in the presence of lpr B cells, normal B cells showed little response to an exogenous, T cell-dependent antigen. The data thus indicate that 1pr B cells manifest an intrinsic abnormality which is essential for autoantibody production in the lpr model.
SummaryInterleukin 5 (I1`5) induces proliferation and differentiation of B cells and eosinophils by interacting with its receptor (I1`5R) which consists of two distinct polypeptide chains, c~ and j8 (~/c). Although both I1`5Ro~ and /3c lack a kinase catalytic domain, I1`5 is capable of inducing tyrosine phosphorylation of cellular proteins. We investigated the role of I1`SKcx in tyrosine phosphorylation of molecules involved in I1`5 signal transduction, using an I1`5-dependent early B cell line, Y16 and transfectants expressing intact or mutant I1`5Rc~ together with intact/3c. The results revealed that the transfectants expressing truncated I1`SKcx, which entirely lacks a cytoplasmic domain, together with/3c, showed neither protein-tyrosine phosphorylation nor proliferation in response to I1`5. This confirms that I1`5Rc~ plays a critical role in protein-tyrosine phosphorylation which triggers cell growth. I1`5 stimulation results in rapid tyrosine phosphorylation of/3c and proteins containing Src homology 2 (SH2) and/or SH3 domains such as phosphatidyl-inositol-3 kinase, Shc, Vav, and HS1, suggesting their involvement in I1`5-mediated signal transduction. IL-5 stimulation significantly enhanced activities of Janus 2 and B cell-specific Bruton's tyrosine kinases (JAK2 and Btk) and increased the tyrosine phosphorylation ofJAK2 kinase. These results and recent data on signaling of growth factors taken together, multiple biochemical pathways driven by tyrosine kinases such as JAK2 and Btk are involved in I1`5 signal transduction.
The human promyelocytic leukemia cell line HL-60 can be induced to differentiate toward neutrophils and subsequently die via apoptosis in vitro. In this paper, we investigated the roles of protein-tyrosine kinases (PTKs) in retinoic acid (RA)-induced granulocytic differentiation of HL-60 cells. Accompanying the RA-induced differentiation, activities of src family PTKs Lyn and Fgr became detected and reached a plateau 2 days after the stimulation. The immunoblotting using anti-phosphotyrosine antibody (PY-20) showed that the proteins of 56 and 53 kDa were predominantly tyrosine-phosphorylated at day 2. Adsorption and immunoprecipitation of the cell lysate by specific antibodies evidenced that these phosphotyrosine-containing proteins are Lyn and Fgr PTKs. The degree of both activities and tyrosine phosphorylation of these PTKs was reduced to be minimal at day 5 when the HL-60 cells start to die by apoptosis. The inhibitors of PTKs, herbimycin A and genistein, were demonstrated to cause premature cell death of HL-60 cells in the presence of RA. The death was the consequence of an apoptotic process. The Ra-treated HL-60 cells, when incubated with specific c-lyn or c-fgr antisense oligodeoxynucleotide, also underwent premature death at day 2. These data implicate that Lyn and Fgr PTKs prevent programmed cell death to promote granulocytic differentiation of HL-60 cells.
We established Jurkat transfectants that overexpress Pyk2 or its mutants, K457A (lysine 457 was mutated to alanine), Pyk2-Y402F (tyrosine 402 to phenylalanine), and Pyk2-Y881F to investigate the role of Pyk2 in T cell activation. Pyk2 as well as kinase-inactive Pyk2-K457A, was phosphorylated at tyrosine residues 402, 580, and 881 upon T cell antigen receptor cross-linking, indicating that these residues are phosphorylated by other tyrosine kinase(s). However, no tyrosine phosphorylation of Pyk2-Y402F was detected while more than 60% of the tyrosine phosphorylation was observed in Pyk2-Y881F. Pyk2-Y402F inhibited the activation of endogenous Pyk2. The degree of activation of both c-Jun NH 2 -terminal kinase and p38 mitogen-activated protein kinase but not extracellular signal-regulated protein kinase after concurrent ligation of T cell antigen receptor and CD28 was reduced by more than 50% in the clones expressing Pyk2-Y402F. Consistent with this inhibition, IL-2 production was significantly diminished in the Pyk2-Y402F-expressing clones. Furthermore, we found that Pyk2, when overexpressed, associates with Zap70 and Vav. Taken together, these findings suggest that Pyk2 is involved in the activation of T cells through its tyrosine 402. Engagement of T cell antigen receptor (TCR)1 by antigen or ligation of TCR by anti-CD3 Ab rapidly activates the Src family protein-tyrosine kinases (PTKs), Fyn and Lck, leading to the phosphorylation of the immunoreceptor tyrosine-based activation motifs in the CD3 complex (1). Phosphorylated immunoreceptor tyrosine-based activation motifs interact with the Src homology (SH) 2 domains of the Syk/Zap70 family PTKs, resulting in the activation of these PTKs, and in turn, in the phosphorylation of an array of cellular proteins (1). Phospholipase C-␥1, phosphatidylinositol 3-kinase, Ras GTPase-activating protein, Vav, Slp76, and LAT (linker for activation of T cells) are tyrosine-phosphorylated early in the activation of the T cells. However, these signals are not sufficient, and additional signals provided by the occupancy of auxiliary receptors (co-receptors), such as CD28, by ligands on the surface of the antigen-presenting cells are necessary (2). Engagement of these receptors triggers pathways that are integrated to induce transcription of the gene for interleukin-2 (IL-2), a major T cell growth factor. Absence of costimulation leads to an anergic state in which T cells lose the ability to induce IL-2 upon restimulation. Production of IL-2 by T cells requires activation of three distinct mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK), c-Jun NH 2 -terminal kinase (JNK) and p38 MAP kinase (3-5). In T cells, JNK and p38 MAP kinase are synergistically activated by costimulation of the TCR/CD3 and CD28 receptors, while no synergy is observed in the ERK activation (4, 5). Although these MAP kinases are supposed to be regulated by different PTKs and small GTPases (6), the more precise mechanisms for the activation of these MAP kinases, especially of J...
Mice with the C3H background show greater behavioral propensity for schizophrenia, including lower prepulse inhibition (PPI), than C57BL/6 (B6) mice. To characterize as‐yet‐unknown pathophysiologies of schizophrenia, we undertook proteomics analysis of the brain in these strains, and detected elevated levels of Mpst, a hydrogen sulfide (H2S)/polysulfide‐producing enzyme, and greater sulfide deposition in C3H than B6 mice. Mpst‐deficient mice exhibited improved PPI with reduced storage sulfide levels, while Mpst‐transgenic (Tg) mice showed deteriorated PPI, suggesting that “sulfide stress” may be linked to PPI impairment. Analysis of human samples demonstrated that the H2S/polysulfides production system is upregulated in schizophrenia. Mechanistically, the Mpst‐Tg brain revealed dampened energy metabolism, while maternal immune activation model mice showed upregulation of genes for H2S/polysulfides production along with typical antioxidative genes, partly via epigenetic modifications. These results suggest that inflammatory/oxidative insults in early brain development result in upregulated H2S/polysulfides production as an antioxidative response, which in turn cause deficits in bioenergetic processes. Collectively, this study presents a novel aspect of the neurodevelopmental theory for schizophrenia, unraveling a role of excess H2S/polysulfides production.
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