有村 裕

Background: Psychological stress affects the immune system. Upon stress occurrence, glucocorticoid is released that binds to the glucocorticoid receptor and regulates gene expression. Thus, we aimed to examine the stress-induced immunomodulatory mechanisms by investigating the expression patterns of stress-inducible genes in murine immune cells.Methods: BALB/c, C57BL/6, glucocorticoid-receptor congenic mice, and corticotropin-releasing hormone (CRH)-deficient mice were exposed to synthetic glucocorticoid, dexamethasone, or placed under a restraint condition. The expression level of stress-related genes, such as Rtp801, Gilz, Mkp-1, Bnip3, and Trp53inp1 was measured in the immune cells in these mice.Results: Short restraint stress induced Rtp801 and Gilz expressions that were higher in the spleen of BALB/c mice than those in C57BL/6 mice. Mkp-1 expression increased equally in these two strains, despite the difference in the glucocorticoid level. These three genes induced by short restraint stress were not induced in the CRH-deficient mice. In contrast, Bnip3 and Trp53inp1 were only upregulated upon longer restraint events. In the thymus, Trp53inp1 expression was induced upon short restraint stress, whereas Gilz expression constantly increased upon short and repetitive restraint stresses.Conclusion: These results suggest that singular and repetitive bouts of stress lead to differential gene expression in mice and stress-induced gene expression in thymocytes is distinct from that observed in splenocytes. Gilz, Rtp801, and Mkp-1 genes induced by short restraint stress are dependent on CRH in the spleen.

Cell proliferation assays are basic and essential techniques for assessing cellular function. Various colorimetric assays, such as MTT-, WST-1-, and resazurin-based assays, are available; however, studies directly comparing the suitability of each method for immune cell proliferation are scarce. Thus, we aimed to determine the best reagent and its optimal conditions based on variables such as cell number range, stimulation dose, kinetics, and compatibility with the cell division assay using CFSE fluorescence dye which is able to directly monitor divided cells by flow cytometry. In the absence of stimulation, MTT solubilized with SDS (MTT-SDS) and resazurin appeared to accurately reflect the cell numbers in a linear fashion. On the other hand, WST-1 exhibited a higher stimulation index following strong stimulation, whereas MTT-SDS and resazurin exhibited a better sensitivity to weak stimulation. A longer duration for stimulation did not necessarily increase sensitivity. CFSE staining revealed incremental cell division in response to anti-CD3 antibody stimulation in a dose-dependent manner. The cell numbers indirectly estimated from cell division profiles were consistent with the dose-response curve in the absorbance of MTT-SDS and resazurin. The absorbance does not increase before cell division, irrespective of T cell activation status, suggesting that these reagents reflect the cell number but not the cellular volume. Collectively, resazurin and MTT-SDS seem to be more reliable than others, and thus appear applicable in various conditions for the immune cell experiments.

T cell activation is regulated by a balance between phosphorylation and dephosphorylation that is under the control of kinases and phosphatases. Here, we examined the role of a non-receptor-type protein tyrosine phosphatase, PTP-PEST, using retrovirus-mediated gene transduction into murine T cells. Based on observations of vector markers (GFP or Thy1.1), exogenous PTP-PEST-positive CD4(+) T cells appeared within 2 days after gene transduction; the percentage of PIP-PEST-positive cells tended to decrease during a resting period in the presence of IL-2 over the next 2 days. These vector markers also showed much lower expression intensities, compared with control cells, suggesting a correlation between the percent reduction and the low marker expression intensity. A catalytically inactive PIP-PEST mutant also showed the same tendency, and stepwise deletion mutants gradually lost their ability to induce the above phenomenon. On the other hand, these PIP-PEST-transduced cells did not have an apoptotic phenotype. No difference in the total cell numbers was found in the wells of a culture plate containing VEC- and PIP-PEST-transduced T cells. Moreover, serine/threonine kinase Akt, but not the anti-apoptotic molecules Bc1-2 and Bcl-XL, reversed the phenotype induced by PTP-PEST. We discuss the novel mechanism by which Ala interferes with PIP-PEST. (C) 2014 Elsevier Inc. All rights reserved.

The aim of the present study was to determine the correlations between leukocyte cell-derived chemotaxin 2 (LECT2) and inflammation-related variables in human inflammatory disease. Plasma samples from 23 septic patients who had been admitted to the intensive care unit (ICU) of our institution and 31 volunteers were used. Plasma LECT2 concentrations were examined retrospectively and compared with those of various inflammatory cytokines and routine laboratory data. The LECT2 concentrations of the septic patients at the time of ICU entry (5.3 +/- 4.1 ng/mL) were significantly lower than those of the volunteers (19.7 +/- 3.4 ng/mL) and these concentrations had significantly increased by the time of ICU discharge. Individual analyses showed that the LECT2 concentrations of all 19 patients had increased by the time of ICU discharge. A combination of LECT2 and C-reactive protein (CRP) concentrations was capable of discriminating the acute and recovery phases of sepsis to a degree similar to those of the combinations of CRP concentration and percentage of neutrophils, CRP concentration and percentage of immature white blood cells, or CRP and interleukin-6 concentrations. Thus, the LECT2 concentration correlates with the severity of systemic inflammation in patients with sepsis. LECT2 may be a reliable diagnostic indicator of human inflammatory diseases.

To elucidate whether leukocyte cell derived chemotaxin 2 (LECT2) controls the progression of staphylococcal enterotoxin A (SEA) induced toxicity, we examined the role of LECT2 in a mouse model Almost all the C57BL/6 J (B6) mice survived for 72 h after the injection of 0 1 mu g of SEA and 20 mg of D galactosamme (D GalN) However, the same treatment protocol in LECT2(-/-) mice produced a high lethality (similar to 90%), severe hepatic apoptosis, and massive hepatic and pulmonary hemorrhage, similar to the situation observed in B6 mice treated with 1 0 mu g SEA/D GalN The plasma LECT2 levels in B6 mice treated with 1 0 mu g SEA/D GalN were inversely correlated with the plasma cytokine levels and were associated with prognosis LECT2 administration increased the survival of B6 mice and down regulated TNF alpha and IL 6 These results suggest the involvement of LECT2 in the regulation of fatal SEA induced toxicity in D GalN sensitized mice (C) 2010 Elsevier Inc All rights reserved

The phosphorylation and dephosphorylation of signaling molecules play a crucial role in various cellular processes, including immune responses. To date, the global expression profile of protein tyrosine phosphatases (PTPs) in various immune cells has not been described. With the RefDIC (Reference Genomics Database of Immune Cells) database compiled by RIKEN (Rikagaku Kenkyusho), we examined the expression patterns of PTP-encoding genes in mice and identified between 57 and 64 PTP-encoding genes (depending on cutoff values) that were commonly expressed in immune cells. Cells of different lineages contained additional, unique PTP-encoding genes, which resulted in a total of 58 to 76 genes. Compared with cells from nonimmune tissues, immune cells exhibited enhanced expression of the genes encoding 8 PTP-encoding genes, including Ptprc, Ptpn6, and Ptpn22, but had barely detectable expression of 11 PTP-encoding genes, including Ptprd and Tns1. Each immune cell lineage had between 2 and 18 PTP-encoding genes expressed at relatively high or low extents relative to the average expression among immune cells; for example, Ptprj in B cells, Dusp3 in macrophages, Ptpro in dendritic cells, and Ptprg in mast cells. These PTPs potentially play important roles in each cell lineage, and our analysis provides insight for future functional studies.

Bacterial infection has become a focus of attention in the pathogenesis of primary biliary cirrhosis (PBC). We earlier reported that the bacterial lipoteichoic acid was detected at the sites of inflammation around damaged bile ducts in the livers of PBC, and PBC patients' sera showed high titers against streptococcal histone-like protein. Here, we investigated whether chronic bacterial exposure could trigger PBC-like epithelial cell damage in normal mouse. BALB/c mice were repeatedly inoculated with various bacteria for 8 weeks. At 1 week (Group 1) and 3, 4, or 20 months (long term; Group 2) after the final inoculation, mice were killed to obtain samples. In the livers of the Streptococcus intermedius (S.i.)-inoculated mice in Group 1, cellular infiltration was predominantly observed around the bile ducts over the hepatic parenchyma. In the S.i.-inoculated mice in Group 2, portal but not parenchymal inflammation was observed in the livers, and periductal cellular infiltrates were detected in the salivary glands. Both S. i.-inoculated Groups 1 and 2 BALB/c mice sera had antibodies against HuCCT1 biliary epithelial cells, anti-nuclear antibodies, and anti-gp210 antibodies, but not anti-mitochondrial antibodies. Immunoreactivity to histone-like DNA-binding protein of S.i. (S.i.-HLP) was detectable around the sites of chronic nonsuppurative destructive cholangitis in the portal area in the livers of both S. i.-inoculated Groups 1 and 2 BALB/c mice. Furthermore, anti-S.i.-HLP antibody bound to synthetic gp210 peptide, as well. Bacteria triggered PBC-like cholangitis, multifocal epithelial inflammation, and autoantibody production. Bacteria are likely involved in the pathogenesis of PBC and of associated multifocal epithelial inflammation. Laboratory Investigation (2010) 90, 577-588; doi: 10.1038/labinvest.2010.40; published online 8 February 2010

PI3K plays crucial roles in the immune system. Mice deficient for p85 alpha, a major regulatory subunit of class IA PI3K, show various defects and alterations in B cells, mast cells, macrophages, and DCs, and peripheral T cells are reportedly normal, at least in vitro. In normal mice, long-term exposure to a SAg, SEA, in vivo induced a high level of the protracted expansion of SEA-reactive V beta 3(+)CD4(+) T cells, whereas the same treatment induced T cell expansion in p85 alpha-deficient mice but to a much lesser extent than in normal mice. However, mixed bone marrow chimera mice, which have normal and p85 alpha-deficient T and B cells, demonstrated equal responses of both T cells following stimulation with a SEA pump. In reciprocal cotransfer experiments of T and B cells from normal and p85 alpha-deficient mice into Rag2-deficient mice, followed by SEA stimulation, p85 alpha-deficient T cells revealed much higher proliferative capacity in the presence of normal B cells than did normal T cells with p85 alpha-deficient B cells. Histologically, a marked B cell reduction was observed in the follicles and MZ of the spleen, and DCs accumulated in the MZ. In addition, p85 alpha-deficient B cells had a low level of MHC class II expression. Collectively, these data suggested that the PI3K p85 alpha subunit alters the SAg presentation capacity of B cells and indirectly modulates the magnitude of the T cell response, which may affect the protection against SEA-containing bacteria. J. Leukoc. Biol. 87: 493-500; 2010.

Loss of VHR phosphatase causes cell cycle arrest in HeLa carcinoma cells, suggesting that VHR inhibition may be a useful approach to halt the growth of cancer cells. We recently reported that V H R is upregulated in several cervix cancer cell lines as well as in carcinomas of the uterine cervix. Here we report the development of multidentate small-molecule inhibitors of VHR that inhibit its enzymatic activity at nanomolar concentrations and exhibit antiproliferative effects on cervix cancer cells. Chemical library screening was used to identify hit compounds, which were further prioritized in profiling and kinetic experiments. SAR analysis was applied in the search for analogs with improved potency and selectivity, resulting in the discovery of hovel inhibitors that are able to interact with both the phosphate-binding pocket and several distinct hydrophobic regions within VHR's active site. This multidentate binding mode was confirmed by X-ray crystallography. The inhibitors decreased the proliferation of cervix cancer cells, while growth of primary normal keratinocytes was not affected. These compounds may be a starting point to develop drugs for the treatment of cervical cancer.

The PTEN tumour suppressor gene is induced by the early growth response 1 (EGR1) transcription factor, which also transactivates p53, p73, and p300/CBP as well as other proapoptotic and anti-cancer genes. Here, we describe a novel Akt-EGR1-alternate reading frame (ARF)-PTEN axis, in which PTEN activation in vivo requires p14ARF-mediated sumoylation of EGR1. This modification is dependent on the phosphorylation of EGR1 at S350 and T309 by Akt, which promotes interaction of EGR1 with ARF at K272 in its repressor domain by the ARF/Ubc9/SUMO system. EGR1 sumoylation is decreased by ARF reduction, and no EGR1 sumoylation is detected in ARF(-/-) mice, which also exhibit reduced amounts of PTEN. Our model predicts that perturbation of any of the clinically important tumour suppressors, PTEN, EGR1, and ARF, will cause some degree of dysfunction of the others. These results also explain the known negative feedback regulation by PTEN on its own synthesis through PI3 kinase inhibition.

We report that the protein tyrosine phosphatase PTP-PEST is expressed in resting human and mouse CD4(+) and CD8(+) T cells, but not in Jurkat T leukemia cells, and that PTP-PEST protein, but not mRNA, was dramatically downregulated in CD4(+) and CD8(+) primary human T cells upon T cell activation. This was also true in mouse CD4(+) T cells, but less striking in mouse CD8+ T cells. PTP-PEST reintroduced into jurkat at levels similar to those in primary human T cells, was a potent inhibitor of TCR-induced transactivation of reporter genes driven by NFAT/AP-1 and NF-kappa B elements and by the entire IL-2 gene promoter. Introduction of PTP-PEST into previously activated primary human T cells also reduced subsequent IL-2 production by these cells in response to TCR and CD28 stimulation. The inhibitory effect of PTP-PEST was associated with dephosphorylation the Lck kinase at its activation loop site (Y394), reduced early TCR-induced tyrosine phosphorylation, reduced ZAP-70 phosphorylation and inhibition of MAP kinase activation. We propose that PTP-PEST tempers T cell activation by dephosphorylating TCR-proximal signaling molecules, such as Lck, and that down-regulation of PTP-PEST may be a reason for the increased response to TCR triggering of previously activated T cells. (C) 2008 Elsevier Ltd. All rights reserved.

Protein tyrosine phosphatases (PTPs) are important regulators of many cellular functions and a growing number of PTPs have been implicated in human disease conditions, such as developmental defects, neoplastic disorders, and immunodeficiency. Here, we review the involvement of PTPs in human autoimmunity. The leading examples include the allelic variant of the lymphoid tyrosine phosphatase (PTPN22), which is associated with multiple autoimmune diseases, and mutations that affect the exon-intron splicing of CD45 (PTPRC). We also find it likely that additional PTPs are involved in susceptibility to autoimmune and inflammatory diseases. Finally, we discuss the possibility that PTPs regulating the immune system may serve as therapeutic targets.

Src family kinases are suppressed by a "tail bite" mechanism, in which the binding of a phosphorylated tyrosine in the C terminus of the protein to the Src homology (SH)2 domain in the N-terminal half of the protein forces the catalytic domain into an inactive conformation stabilized by an additional SH3 interaction. In addition to this intramolecular suppressive function, the SH2 domain also mediates intermolecular interactions, which are crucial for T cell antigen receptor (TCR) signaling. To better understand the relative importance of these two opposite functions of the SH2 domain of the Src family kinase Lck in TCR signaling, we created three mutants of Lck in which the intramolecular binding of the C terminus to the SH2 domain was strengthened. The mutants differed from wild-type Lck only in one to three amino acid residues following the negative regulatory tyrosine 505, which was normally phosphorylated by Csk and dephosphorylated by CD45 in the mutants. In the Lck-negative JCaM1 cell line, the Lck mutants had a much reduced ability to transduce signals from the TCR in a manner that directly correlated with SH2-Tyr(P)(505) affinity. The mutant with the strongest tail bite was completely unable to support any ZAP-70 phosphorylation, mitogen-activated protein kinase activation, or downstream gene activation in response to TCR ligation, whereas other mutants had intermediate abilities. Lipid raft targeting was not affected. We conclude that Lck is regulated by a weak tail bite to allow for its activation and service in TCR signaling, perhaps through a competitive SH2 engagement mechanism.

The long-term exposure of mice to superantigen SEA using a mini-osmotic pump (SEA pump) induced a long-lasting expansion of V beta 3(+)CD4(+) T cells with T helper (Th) 2 cell-type properties. Removal of the SEA pump 10 days after pump implantation did not significantly alter the level of V beta 3(+)CD4(+) T cell expansion/maintenance. Furthermore, CFSE-labeled CD4+ T cells failed to divide when transferred to post-implantation day 15 mice. Thus, CD4+ T cells appeared to survive for at least 30 days in the absence of a sufficient amount of antigen to trigger cell division. STAT6 deficient mice, in which Th2 cell development is largely impaired, also exhibited a protracted cell expansion, similar to that observed in normal mice, suggesting that the Th2 cell property is dispensable for the maintenance of V beta 3(+)CD4(+) T cell expansion. The expanded CD4+ T cells on post-implantation day 26 were arrested in the G(0)/G(1) phase of the cell cycle and showed a lower level of cell division upon restimulation. The Cdk inhibitor p27(Kip1) was highly expressed, and Cdk2 was downregulated. Moreover, the CD4+ T cells were resistant to in vitro apoptosis induction in parallel with their level of Bcl-2 expression. Collectively, the V beta 3(+)CD4(+) T cells appeared to develop into long-lived memory T cells with cell cycle arrest upon long-term exposure to SEA. (C) 2007 Elsevier Inc. All rights reserved.

PTPN3 (PTPH1) is a cytoskeletal protein tyrosine phosphatase that has been implicated as a negative regulator of early TCR signal transduction and T cell activation. To determine whether PTPN3 functions as a physiological negative regulator of TCR signaling in primary T cells, we generated gene-trapped and gene-targeted mouse strains that lack expression of catalytically active PTPN3. PTPN3 phosphatase-negative mice were born in expected Mendelian ratios and exhibited normal growth and development. Furthermore, numbers and ratios of T cells in primary and secondary lymphoid organs were unaffected by the PTPN3 mutations and there were no signs of spontaneous T cell activation in the mutant mice with increasing age. TCR-induced signal transduction, cytokine production, and proliferation was normal in PTPN3 phosphatase-negative mice. This was observed using both quiescent T cells and recently stimulated T cells where expression of PTPN3 is substantially up-regulated. We conclude, therefore, that the phosphatase activity of PTPN3 is dispensable for negative regulation of TCR signal transduction and T cell activation.

To enhance diagnostic or therapeutic efficacy, novel nanomaterials must be engineered to function in biologically relevant environments, be visible by conventional fluorescent microscopy, and have multivalent loading capacity for easy detection or effective drug delivery. Here we report the fabrication of silica nanoparticles doped with quantum dots and superficially functionalized with amino and phosphonate groups. The amino groups were acylated with a water-soluble biotin-labeling reagent. The biotinylated nanoparticles were subsequently decorated with neutravidin by exploiting the strong affinity between neutravidin and biotin. The resultant neutravidin-decorated fluorescent silica nanoparticles stably dispersed under physiological conditions, were visible by conventional optical and confocal fluorescent microscopy, and could be further functionalized with macromolecules, nucleic acids, and polymers. We also coated the surface of the nanoparticles with biotinylated mouse anti-human CD3 (alpha CD3). The resultant fluorescent nanoassembly was taken up by Jurkat T cells through receptor-mediated endocytosis and was partially released to lysosomes. Thus, quantum dot-doped silica nanoparticles decorated with neutravidin represent a potentially excellent scaffold for constructing specific intracellular nanoprobes and transporters.

Comparative studies using T(h)2-prone BALB/c and T(h)1-prone C57BL/6 mice were performed to clarify the influence of genetic background on T-h cell differentiation. The results showed IL-4, the production of which is induced by IL-2, to be much more abundantly produced by BALB/c naive CD4(+) T cells than by C57BL/6 naive CD4(+) T cells, thereby leading to a tendency for differentiation toward T(h)2 in BALB/c naive CD4(+) T cells. This difference in IL-4 production between the two naive CD4(+) T cells appeared to be attributable to specific intracellular signaling events. Signal transducer and activator of transcription 5 (STAT5) was preferentially activated by IL-2 in CD4(+) T cells developing in BALB/c in contrast to the corresponding cells in C57BL/6. In addition, IL-4 also induced stronger STAT5 activation in CD4(+) T cells developing in BALB/c than in those developing in C57BL/6, whereas STAT6 was equally activated in these two cells. Further results supported the involvement of STAT5 in the difference in T-h cell differentiation between BALB/c and C57BL/6 naive CD4(+) T cells. STAT5A(-/-) naive CD4(+) T cells with the BALB/c genetic background showed markedly less IL-2-induced IL-4 production than BALB/c naive CD4(+) T cells. Conversely, forced expression of the constitutively active forms of STAT5A and STAT5B in C57BL/6 naive CD4(+) T cells promoted the differentiation of T(h)2 cells. Thus, our results indicate IL-2-induced IL-4 production by naive CD4(+) T cells, in which STAT5 activation is involved and directly controlled by the genetic background, to influence T-h cell differentiation in murine strains.

We previously reported that V beta 3(+) CD4(+) T cells maintained a protracted expansion, with the phenotypes of memory Th2 cells, for 30 days in C57BL/6 (B6) mice implanted with SEA-containing mini-osmotic pumps. In the present Study, we followed the fate of V beta 3(+) CD4(+) T cells in CD28(-/-) mice. V beta 3(+) CD4(+) T cells increased to a degree similar to that of B6 V beta 3(+) CD4(+) T cells until day 10 after implantation, then declined rapidly reaching the control level by 28 days. Remaining V beta 3(+) CD4(+) T cells at that time did not exhibit memory phenotypes nor Th2-deviated responses. The rapid drop in VP3(+) CD4(+) T cells in CD28(-/-) mice was attributable to upregulated induction of apoptosis owing to marginal inductions of Bcl-2 and Bcl-x(L). Collectively, these data indicate CD28 to play critical roles in the generation and maintenance of SEA-reactive CD4(+) T cells in vivo. (c) 2006 Elsevier Inc. All rights reserved.

Both CD28 and its relative, inducible costimulator ( ICOS), have a binding motif for phosphatidylinositol 3-kinase (PI3K) in their cytoplasmic tail, and the binding of PI3K leads to activation of a serine/threonine kinase, Akt. The role of Akt in cytokine production and helper T (Th) cell differentiation remains obscure. In this study, we found that enforced expression of the constitutively active form (E40K) of Akt rendered CD4(+) T cells activated. Wild-type of Akt and E40K promoted Th1 cell differentiation in C57BL/6-derived and Th1-polarized BALB/c-derived CD4(+) T cells, while both promoted Th2 cell differentiation in BALB/c-derived and Th2-polarized C57BL/6 CD4(+) T cells. E40K also facilitated Th1 differentiation in CD4(+) T cells from IL-4-deficient mice with the BALB/c background. E40K up-regulated expression of NF-AT and c-Myb, which may be related to the augmentation of cytokine production by E40K. These findings indicate that the mechanism by which Akt augments cytokine production via CD28 and ICOS is Th cell type-specific and reflects the intracellular status affected by the cytokine milieu. We conclude that Akt is a neutral amplifier of T cell activation and Th differentiation.

To determine the levels of maturation and differentiation of murine CD4 single-positive (SP) T cells, we compared the secondary responses of staphylococcal enterotoxin A (SEA)-induced neonatal thymic, adult thymic and adult splenic CD4 SP T cell blasts prepared from whole or heat-stable antigen(low) CD4 SP T cells. Proliferative responses upon re-stimulation with SEA were strong in adult splenic CD4 SP T cell blasts, but quite weak in neonatal thymic and adult thymic CD4 SP T cell blasts. SEA-induced IL-2 production was weaker in neonatal thymic blasts than in the adult splenic CD4 SP T cell blasts. In contrast, SEA-induced IL-4 production was high in neonatal thymic CD4 SP T cell blasts, and low in adult splenic and thymic CD4 SP T cell blasts. Expression of GATA-3, that directs production of IL-4 in T cells, examined at protein and mRNA levels, was higher in neonatal thymic cells than in adult thymic and splenic cells. These results suggest that neonatal and adult thymic CD4 SP T cells in the final stage of maturation are relatively immature compared with adult splenic CD4 SP T cells. The cytokine production profile of neonatal thymic CD4 SP T cells suggests that they are inclined towards a T(h)2 response.

We found a tight correlation among the levels of H4/inducible costimulator (ICOS) expression, IL-4 production, and GATA-3 induction, using activated CD4(+) T cells obtained from six different murine strains. BALB/c-activated CD4(+) T cells expressed similar to10-fold more H4/ICOS on their surfaces and produced similar to10-fold more IL-4 upon restimulation than C57BL/6-activated CD4(+) T cells. BALB/c naive CD4(+) T cells were shown to produce much higher amounts of IL-2 and IL-4 upon primary stimulation than C57BL/6 naive CD4(+) T cells. Neutralization of IL-4 with mAbs in culture of BALB/c naive CD4(+) T cells strongly down-regulated both H4/ICOS expression on activated CD4(+) T cells and IL-4 production upon subsequent restimulation. Conversely, exogenous IL-4 added to the culture of BALB/c or C57BL/6 naive CD4(+) T cells up-regulated H4/ICOS expression and IL-4 production upon restimulation. In addition, retroviral expression of GATA-3 during the stimulation of naive CD4(+) T cells from C57BL/6 or IL-4(-/-) mice increased H4/ICOS expression on activated CD4(+) T cells. A similar effect of IL-2 in the primary culture of BALB/c naive CD4(+) T cells appeared to be mediated by IL-4, the production of which was regulated by IL-2. These data suggest that IL-4 induced by IL-2 is critical to the maintenance of high H4/ICOS expression on BALB/c-activated CD4(+) T cells.

We examined the co-stimulatory activity of H4/ICOS on murine activated CD4(+) T cells and found that the cross-linking of H4/ICOS enhanced their proliferation, in addition to raising IFN-gamma, IL-4 and IL-10 production to levels comparable to those induced by CD28. However, IL-2 production was only marginally co-stimulated by H4/ICOS. This distinct pattern of lymphokine production appears to be induced by a specific intracellular signaling event. Compared with CD28, H4/ICOS dominantly elicited the Akt pathway via phosphatidylinositol 3-kinase. In addition, mitogen-activated protein kinase family kinases were activated in different ways by CD28 and H4/ICOS. The strong phosphorylation of p46 c-Jun N-terminal kinase was observed upon CD28 co-stimulation, but was less potently induced by H4/ICOS. The strain diversity in the induction of H4/ICOS was recognized. The expression of H4/ICOS on BALB/c activated CD4(+) T cells was >6-fold higher compared with C57BL/6 activated CD4(+) T cells. Furthermore, BALB/c activated CD4(+) T cells exhibited more T(h)2-deviated lymphokine production as compared with C57BL/6 activated CD4(+) T cells and signaling through H4/ICOS during the primary stimulation of naive CD4(+) T cells promoted the generation of T(h)2 cells. Thus, the difference in H4/ICOS expression on activated CD4(+) T cells, which is regulated among the mouse strains, may also regulate the polarization of T-h cells.

The behavior of Vβ2+ T cells reactive with the pathogenic superantigen toxic shock syndrome toxin-1 (TSST-1) was examined in 12 patients with neonatal TSS-like exanthematous disease (NTED). In 8 patients, the percentage of Vβ2+ T cells in peripheral blood (PB) T cells had increased to three times the level in methicillin-resistant Staphylococcus aureus (MRSA)-free neonatal controls, when examined on days 4 to 7 of life. In the remaining 4 patients, however, the percentage of Vβ2- T cells was significantly low, i.e., ranging from less than 10% to 30% of the control level, when they were examined on days 4 to 8 of life, and the percentage increased dramatically several days later, indicating that these patients actually had NTED. We recommend that, in patients who are suspected of having NTED, when an increase in Vβ2+ T cells is not observed in a comparatively early period of the illness, reassessment of the Vβ2+ T cells should be carried out 2 to 4 days later, to obtain a conclusive diagnosis of NTED.

Stimulation of B cell antigen receptor (BCR) may induce proliferation, differentiation, or apoptosis, depending upon the maturational stage of the cell and the presence or absence of signals transmitted via coreceptors. One such signal is delivered via CD40; for instance, ligation of CD40 rescues B cells from BCR-induced apoptosis. Here we show that, in contrast to WEHI-231 cells, CD40 ligation did not reverse BCR-induced growth inhibition in the BAL-17 mature B cell line and CD40 ligation itself inhibited proliferation. This inhibitory signaling was not observed in CD45-deficient cells. Further analyses demonstrate that transfection of dominant-negative form of SEK1 or treatment with SB203580 strongly reduced CD40-induced inhibition of BAL-17 proliferation, suggesting a requirement for c-Jun NH2-terminal kinase and p38 in CD40-induced inhibition of proliferation. Interestingly, CD40-initiated activation of c-Jun NH2-terminal kinase and p38 was enhanced and sustained in CD45-deficient cells, and these phenotypes were reversed by transfecting CD45 gene. However, CD40-mediated induction of cell surface molecules was not affected in CD45-deficient cells. Taken collectively, these results suggest that CD45 exerts a decisive effect on selective sets of CD40-mediated signaling pathways, dictating B cell fate.

In this study, we examined the contribution made by CD45 to B cell antigen receptor (BCR)-induced activation of mitogen-activated protein kinase (MAPK) family members, We found that CD45 negatively regulated BCR-induced c-Jun NH2-terminal kinase (JNK) and p38 activation in immature WEHI-231 cells, whereas in mature BAL-17 cells, CD45 positively regulated JNK and p38 activation and negatively regulated extracellular signal-regulated kinase activity. Furthermore, cooperative action of JNK and p38 dictated BCR-induced inhibition of growth. Thus, CD45 appears to differentially regulate BCR-induced activation of MAPK members, and can exert opposing effects on JNK and p38 in different cellular milieu, controlling the B cell fate. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Src homology region 2 (SH2) domain-containing phosphatase-1 (SXIP-1) is a cytosolic protein tyrosine phosphatase containing two SH2 domains in its NH2 terminus. That immunological abnormalities of the motheaten and viable motheaten mice are caused by mutations in the gene encoding SHP-1 indicates that SHP-1 plays important roles in lymphocyte differentiation, proliferation, and activation. To elucidate molecular mechanisms by which SHP-1 regulates BCR-mediated signal transduction, we determined SHP-1 substrates in B cells using the substrate-trapping approach. When the phosphatase activity-deficient form of SHP-1, in which the catalytic center cysteine (C453) was replaced with serine (SHP-1-C/S), was introduced in WEHI-231 cells, tyrosine phosphorylation of a protein of about 70 kDa was strongly enhanced. Immunoprecipitation and Western blot analyses revealed that this protein is the B cell linker protein (BLNK), also named SH2 domain leukocyte protein of 65 kDa, and that upon tyrosine phosphorylation BLNK binds to SHP-1-C/S in vitro. In vitro kinase assays demonstrated that hyperphosphorylation of BLNK in SHP-1-C/S-expressing cells was not due to enhanced activity of Lyn or Syk, Furthermore, BCR-induced activation of c-Jun NH2-terminal kinase was shown to be significantly enhanced in SHP-1-C/S transfectants. Taken collectively, our results suggest that BLNK is a physiological substrate of SHP-1 in B cells and that SHP-1 selectively regulates c-Jun NH2-terminal kinase activation.

Using CD-15-deficient clones from the immature B cell line, WEHI-231, we previously demonstrated that CD45 selectively dephosphorylates the Src-family protein tyrosine kinase Lyn and inhibits its kinase activity. To further define the mechanisms of CD45 action on Lyn, we metabolically labeled Lyn from CD45-positive and -negative WEHI-231 cells and analyzed cyanogen bromide fragments by SDS-PAGE analysis. Phosphoamino acid analysis confirmed that Lyn is tyrosine phosphorylated with little serine or threonine phosphorylation. In CD45-negative cells, two bands at 8.2 and 4.1 kDa were phosphorylated in the absence of B cell Ag receptor (BCR) ligation. The 8.2-kDa band corresponded to a fragment containing the positive regulatory site (Tyr(397)), as assessed by its size and its phosphorylation in an in vitro kinase assay, The 4.1-kDa band was phosphorylated by COOH-terminal terminal Src kinase, suggesting that it contains the COOH-terminal negative regulatory site (Tyr(508)). CD45 was also shown to dephosphorylate autophosphorylated Lyn in vitro. Thus, CD45 dephosphorylates not only the negative but also the positive regulatory tyrosine residues of Lyn, Furthermore, coimmunoprecipitations using anti-Ig alpha Ab demonstrated that Lyn associated with the resting BCR was constitutively phosphorylated and activated in CD45-negative cells, In the parental cells, both regulatory sites were phosphorylated on BCR ligation, Taken collectively, these results suggest that CD45 keeps both BCR-associated and total cytoplasmic pools of Lyn in an inactive state, and a mechanism by which Lyn is activated by relative reduction of CD45 effect may be operative on BCR ligation.

Signaling events leading to B cell growth or apoptosis are beginning to be unravelled, but detailed information is still lacking. To identify signaling molecules involved in B cell antigen receptor (BCR)-initiated pathways, we used the immature B cell line, WEHI-231, to investigate protein tyrosine phosphatases (PTP) whose expression was modulated by BCR ligation. Among the PTP cloned by reverse transcription-PCR, mRNA expression of the proline-, glutamic acid-, serine- and threonine-rich (PEST) domain phosphatase (PEP) was selectively elevated 3.1-fold within 3 h after anti-IgM antibody stimulation. In contrast, expression of another PEST domain phosphatase, PTP-PEST, was unaffected. Western blot analysis revealed that 71 % of PEP was located in the cytosolic fraction, while 29 % was in the membrane fraction. To examine the direct contribution made by PEP to BCR-initiated signal transduction, we transfected an antisense PEP cDNA into WEHI-231 cells. Two stable clones were established in which PEP expression was reduced by 34 % and 47 %, respectively. Strikingly, BCR-mediated inhibition of DNA synthesis was significantly rescued in the clones, and G1 phase cell cycle arrest acid apoptosis were almost completely ablated. Considered collectively,these results indicate that PEP is a positive, crucial regulator in determining B cell fate triggered by BCR engagement.

We previously reported the cloning of a human S10 cDNA which encodes a small GTP-binding protein belonging to the Rab subfamily. Here we describe a mouse S10 cDNA and its genomic structure. Mouse S10 is 92.3% homologous at the nucleotide level and 98.3% identical at the amino acid level compared to human SIG. The mouse S10 gene is comprised of two exons and a single intron. Northern blotting of tissue RNAs indicates that the S10 gene is predominantly expressed in brain. (C) 1997 Elsevier Science B.V.

A cDNA fragment of the Borna disease virus (BDV) open reading frame II (ORF-II), which encodes a 24-kDa phosphoprotein (p24 [P protein]), was amplified from total RNA of peripheral blood mononuclear cells (PBMC) from three psychiatric inpatients. The amplified cDNA fragments mere cloned, sequenced, and analyzed. A total of 15 clones, 5 from each patient, were studied. Intrapatient divergencies of the BDV ORF-II nucleotide sequence were 4.2 to 7.3%, 4.8 to 7.3%, and 2.8 to 7.1% for the three patients, leading to differences of 7.7 to 14.5%, 10.3 to 17.1%, and 6.0 to 16.2%, respectively, in the deduced amino acid sequence for BDV p24. Interpatient divergencies among the 15 clones were 5.9 to 12.7% at the nucleotide level and 12.8 to 28.2% at the amino acid level. Thus, in p24, BDV in human PBMC of the patients undergoes mutation at high rates in vivo. Additionally, we found that the nucleotide Sequence of the 15 human BDV ORF-II cDNA clones differed from those of the horse strains V and He/80-1 by 4.2 to 9.3%. However, comparison of the consensus amino acid sequence deduced from the 15 human clones with those of the horse strains revealed no human-specific amino acid residue, suggesting that the BDV infecting humans may be related to that infecting horses.

The hst-1 gene, which is implicated in mammalian embryonic development and morphological transformation of NIH3T3 cells, is expressed in undifferentiated F9 cells, but not in differentiated F9 and other well-differentiated cells, such as PYS-2, NIH3T3 and HeLa cells. An octamer element present in the 3' untranslated region acts as an enhancer. Although Oct3 is down-regulated when F9 cells are differentiated, transient expression of Oct3 did not enhance the hst-1 promoter activity in HeLa, NIH3T3 or PYS-2 cells. Thus, the role of Oct3 on hst-1 expression remains elusive, and an additional transcription factor which interacts may regulate hst-1 transcription in association with Oct1, Oct3 or both.

A genomic HLA-G clone named 7.OE was isolated from a Japanese placenta. The deduced amino acid sequence of the 7.0E was identical to two HLA-G genomic clones and two cDNA clones previously described. The DNA sequences of alpha1 and alpha2 domains of the HLA-G gene from 5 cell lines also encoded the same amino acids. However, a 14 bp insertion, ATTTGTTCATGCCT, was present in the 3' untranslated region of 7.OE compared with the originally described HLA-G clone (HLA 6.0). Polymerase chain reaction (PCR)/single strand conformational polymorphism (SSCP) analysis of exon 8 allowed the HLA-G gene to be classified into two alternative types, G6.0 and 7.0 E, those correlated to the absence or the presence of the 14 bp stretch. Each group had minor sequence variant(s), and the alleles of the 7.OE-type were more heterogeneous than those of the G6.0- type. The 14 bp deletion is present only in the G6.0-type of HLA-G alleles among HLA class I genes.Thus it was suggested that G6.0 alleles were generated after diversification of the HLA-G.