Tyrosine phosphorylation and association with phospholipase C gamma-1 of the GAP-associated 62-kD protein after CD2 stimulation of Jurkat T cell

Numerous substrates are tyrosine phosphorylated upon CD2 stimulation of human Jurkat T cells using a mitogenic pair of CD2 monoclonal antibodies, including the phospholipase C (PLC)gamma-1-p35/36 complex. Most of these substrates are identically tyrosine phosphorylated after CD3 ligation, suggesting that both stimuli share the same biochemical pathway. We show, however, in this report that a 63-kD protein is specifically phosphorylated on tyrosine residues after ligation of the CD2 molecule. The tyrosine phosphorylation of p63 can be induced independently of other substrates when using a single CD2 mAb recognizing the D66 epitope of the molecule. Importantly, this CD2- induced tyrosine phosphorylation of p63 can also occur in the absence of the CD3 zeta chain membrane expression, and is also distinct from the protein tyrosine kinases p56lck and p59fyn. We demonstrate, moreover, that p63 is physically linked with PLC gamma-1 and p35/36 upon CD2 stimulation. Finally, we also show that a 62-kD protein coimmunoprecipitating with the p21ras GTPase activating protein (GAP) is heavily tyrosine phosphorylated only after CD2 stimulation. This ultimately suggests that p63 may represent in fact the 62-kD protein that associates with GAP after tyrosine phosphorylation. Taken together, these results demonstrate the occurrence in Jurkat cells of a tyrosine kinase pathway specifically coupled to the CD2 molecule. They also suggest a function of the p62-GAP-associated protein as a link between PLC gamma-1 and p21ras activation pathways after CD2 activation.     

Association of the adaptor molecule LAT with CD4 and CD8 coreceptors identifies a new coreceptor function in T cell receptor signal transduction

Linker for activation of T cells (LAT) is an adaptor protein whose tyrosine phosphorylation is critical for transduction of the T cell receptor (TCR) signal. LAT phosphorylation is accomplished by the protein tyrosine kinase ZAP-70, but it is not at all clear how LAT (which is not associated with the TCR) encounters ZAP-70 (which is bound to the TCR). Here we show that LAT associates with surface CD4 and CD8 coreceptors and that its association is promoted by the same coreceptor cysteine motif that mediates Lck binding. In fact, LAT competes with Lck for binding to individual coreceptor molecules but differs from Lck in its preferential association with CD8 rather than CD4 in CD4(+)CD8(+) thymocytes. Importantly, as a consequence of LAT association with surface coreceptors, coengagement of the TCR with surface coreceptors induces LAT phosphorylation and the specific recruitment of downstream signaling mediators to coreceptor-associated LAT molecules. These results point to a new function for CD4 and CD8 coreceptors in TCR signal transduction, namely to promote LAT phosphorylation by ZAP-70 by recruiting LAT to major histocompatibility complex-engaged TCR complexes.     

Coclustering CD45 with CD4 or CD8 alters the phosphorylation and kinase activity of p56lck

Antibody-mediated CD4 crosslinking results in increased tyrosine phosphorylation and tyrosine kinase activity of the associated p56lck. Treatment with anti-CD4 and anti-Ig also induced the phosphorylation of p56lck in a CD45- mutant cell line, indicating that the increase in phosphotyrosine content of p56lck is not the result of being sequestered from CD45 protein tyrosine phosphatase (PTPase). Antibody-mediated coclustering of CD45 with CD4 inhibited the anti-CD4-induced phosphorylation of p56lck on tyrosine and the concomitant increase in in vitro kinase activity. Similar results were obtained when CD45 was coclustered with CD8 on cytotoxic T cell lines. These observations provide strong evidence that p56lck is a substrate for CD45 in vivo and provide an assay to study the regulation and specificity of CD45 PTPase activity.     

CD4-mediated stimulation of human eosinophils: lymphocyte chemoattractant factor and other CD4-binding ligands elicit eosinophil migration

Lymphocyte chemoattractant factor (LCF) is a tetrameric glycoprotein of 56,000 relative molecular mass produced by activated T lymphocytes. LCF binds to CD4 and has previously been found to stimulate migration of CD4+ lymphocytes and monocytes. Because human eosinophils, like T cells and monocytes, express CD4, we examined functional responses of eosinophils to LCF. Recombinant LCF (rLCF) expressed in COS cells was purified on a CD4 affinity column. Migration of eosinophils was elicited by rLCF at low concentrations: the 50% effective dose (ED50) was 10(-12) to 10(-11) M, concentrations 100- to 1,000-fold lower than the ED50s for the recognized eosinophil chemoattractants C5a and platelet-activating factor. Two other ligands which bound to CD4, human immunodeficiency virus-1 envelope glycoprotein gp120 and monoclonal antibody OKT4, also stimulated eosinophil migration. Monovalent OKT4 Fab competitively inhibited eosinophil responses to rLCF. rLCF did not influence other functional responses of eosinophils tested, including degranulation, superoxide generation, leukotriene C4 production, in vitro survival, or surface expression of the adherence receptor CR3 (CD11b), human histocompatibility leukocyte antigen DR, or interleukin 2 receptor p55 (CD25). We conclude that CD4 on eosinophils is capable of transducing a migratory stimulus and serves as a receptor for a chemoattractant lymphokine LCF. T cell-derived LCF may contribute to recruitment of eosinophils and CD4+ mononuclear cells concomitantly at inflammatory reactions.     

lnc-Rps4l-encoded peptide RPS4XL regulates RPS6 phosphorylation and inhibits the proliferation of PASMCs caused by hypoxia

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NFATc2 and NFATc3 transcription factors play a crucial role in suppression of CD4+ T lymphocytes by CD4+ CD25+ regulatory T cells

The phenotype of NFATc2(-/-) c3(-/-) (double knockout [DKO]) mice implies a disturbed regulation of T cell responses, evidenced by massive lymphadenopathy, splenomegaly, and autoaggressive phenomena. The population of CD4(+) CD25(+) T cells from DKO mice lacks regulatory capacity, except a small subpopulation that highly expresses glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) and CD25. However, neither wild-type nor DKO CD4(+) CD25(+) regulatory T cells (T reg cells) are able to suppress proliferation of DKO CD4(+) CD25(-) T helper cells. Therefore, combined NFATc2/c3 deficiency is compatible with the development of CD4(+) CD25(+) T reg cells but renders conventional CD4(+) T cells unresponsive to suppression, underlining the importance of NFAT proteins for sustaining T cell homeostasis.     

Tyrosine phosphorylation of an interleukin 2 receptor-like protein in cells transformed by human T cell leukemia virus type I

Three interleukin 2 (IL-2)-independent human T cell lines transformed with human T cell leukemia virus type I were analyzed for o-phosphotyrosine-containing proteins (Ptyr proteins). Membrane and intracellular immunofluorescence was positive with antibody to Ptyr (Ptyr antibody). 10 size classes of Ptyr proteins with molecular masses of 81-28 kD were isolated with Ptyr antibodies. Among these, proteins of 64 kD (pI 4.5 and 4.8-5.3) and 45 kD (pI 4.3) were found on the outer cell surface. The Ptyr protein of 64 kD (pI 4.5) had the characteristics of the IL-2 receptor (IL-2-R) in that it was recognized by two monoclonal antibodies directed against different epitopes on the IL-2-R.     

Quantitative analysis of CD6, CD4 and CD8 cell surface molecules compared to the absolute numbers of CD6+, CD4+ and CD8+ T-cells in peripheral blood in patients with HIV-infection.

T lymphocyte subsets were determined on blood samples from 16 HIV-seropositive patients with manifest AIDS (CDC IV), 24 HIV-seropositive patients with lymphadenopathy syndrome (LAS, CDC III), 16 HIV-seropositive clinical healthy persons (CDC II) and 11 HIV-seronegative homosexuals as control group. Absolute numbers of T-cells (CD6+), T-helper/inducer-cells (CD4+) and T-suppressor/cytotoxic-cells (CD8+), obtained by immunofluorescence staining were compared with the absolute amount of subset specific surface molecules, obtained by a T-cell-ELISA. With both, indirect immunofluorescence technique and ELISA technique a highly significant decrease of the absolute numbers of CD4+ cells and the absolute amount of CD4 surface molecules, respectively, was found in asymptomatic HIV-infection, LAS and in manifest AIDS. In all HIV-seropositive groups the relative decrease of CD4 surface molecules was significantly greater than the decline of CD4+ cells. This phenomenon however was not seen in HIV-seronegative homosexuals. The absolute number of CD6+ cells and the amount of CD6 surface molecules were found significantly lowered in AIDS compared to HIV-seronegative homosexuals. No significant changes were found for CD8+ cell numbers and CD8 surface molecule in the progression of the HIV-infection.     

Engagement of CD4 and CD8 expressed on immature thymocytes induces activation of intracellular tyrosine phosphorylation pathways

Accumulating data suggest that the target cells for selection events leading to establishment of the mature T cell repertoire are the functionally immature double-positive (CD4+/CD8+) thymocytes, and that the CD4 and CD8 antigens expressed on these cells play important roles in these processes. In an attempt to define the biochemical pathways implicated in these events, we have studied the effects of engagement of accessory molecules on tyrosine protein phosphorylation. The results of our experiments demonstrate that engagement of CD4 and CD8 expressed on double-positive thymocytes is coupled with a rapid tyrosine protein phosphorylation signal. Further analyses have revealed that these two surface molecules are physically associated with the internal membrane tyrosine protein kinase p56lck in immature thymocytes, and that the catalytic function of lck expressed in double-positive thymocytes is significantly enhanced upon engagement of CD4. These data provide evidence that tyrosine-specific protein phosphorylation pathways coupled to the CD4 and CD8 T cell surface antigens are functional in immature thymocytes, and therefore, formally prove that signaling functions of CD4 and CD8 molecules are operative in immature thymocytes.     

Role of CD4 molecule in the induction of interleukin 2 and interleukin 2 receptor in class II major histocompatibility complex-restricted antigen-specific T helper clones. T cell receptor/CD3 complex transmits CD4-dependent and CD4-independent signals.

The CD4 molecule plays an essential role in antigen-induced activation of T helper (Th) cells, but its contribution to signal transduction events resulting in physiologic T cell function is ill defined. By utilizing anti-CD4 monoclonal antibodies (MAbs) that recognize distinct epitopes of CD4, we have investigated the role of CD4 molecule in antigen-induced interleukin 2 (IL-2) and IL-2 receptor (IL-2R) alpha chain expression in class II major histocompatibility complex-restricted antigen-specific human Th clones. Pretreatment of the Th clones with Leu3a resulted in a dose-dependent suppression of antigen-induced proliferative responses, inositol phosphate accumulation, increase in free cytoplasmic calcium ions ([Ca2+]i), IL-2 mRNA accumulation, IL-2 secretion, and membrane IL-2R expression. IL-2R mRNA accumulation, however, was unaffected even at highest Leu3a concentrations. Leu3a treatment did not affect bypass activation of T cells with PMA plus ionomycin or activation via CD2 molecule. The MAb OKT4, which binds another domain of CD4, was not inhibitory. These results suggest that after T cell antigen receptor-CD3 activation, IL-2 gene induction, IL-2 secretion, and membrane IL-2R expression are absolutely dependent upon participation of CD4 molecules, phosphatidylinositol (PI) hydrolysis, and increase in [Ca2+]i. The requirement for IL-2R gene induction, however, occurs independently of CD4 molecule participation and PI hydrolysis.     

Role of interleukin 6 for differential responsiveness of naive and memory CD4+ T cells in CD2-mediated activation

The present study was undertaken to elucidate different requirements for CD2-mediated activation of naive (CD45RO-) and memory (CD45RO+) CD4+ T cells. A mitogenic combination of anti-CD2 (anti-T11(2) and anti-T11(3] mAbs could effectively induce the proliferation of memory CD4+ T cells even in the absence of monocytes. In marked contrast, naive CD4+ T cells did not disclose any proliferative responses to anti-CD2 mAbs, when monocytes were absent in culture. This differential responsiveness of naive and memory CD4+ T cells appeared to be related largely to a difference in IL-6-producing ability between both populations. IL-6 among monocyte-derived cytokines could correct unresponsiveness of naive CD4+ T cells to anti-CD2 stimulation. Unlike naive CD4+ T cells, memory CD4+ T cells produced IL-6 by themselves, with its mRNA being expressed on anti-CD2 stimulation. Anti-IL-6R mAb significantly inhibited proliferation of memory CD4+ T cells seen in the anti-CD2-stimulated cultures without monocytes, indicating the involvement of their own production of IL-6 in CD2-mediated activation. The results suggest an essential role of IL-6 for triggering of CD4+ T cells via the CD2 molecule.     

Selective induction of human hepatic cytochromes P450 2B6 and 3A4 by metamizole

The pyrazolone drug metamizole is a widely used analgesic. Analysis of liver microsomes from patients treated with metamizole revealed selectively higher expression of cytochromes P450, CYP2B6 and CYP3A4 (3.8- and 2.8-fold, respectively), and 2.9-fold higher bupropion hydroxylase activity compared with untreated subjects. Further investigation of metamizole and various derivatives on different potential target genes in human primary hepatocytes demonstrated time- and concentration-dependent induction by metamizole of CYP2B6 (7.8- and 3.1-fold for mRNA and protein, respectively, at 100 muM) and CYP3A4 (2.4- and 2.9-fold, respectively), whereas other genes (CYP2C9, CYP2C19, CYP2D6, NADPH:cytochrome P450 reductase, ABCB1, constitutive androstane receptor (CAR), pregnane X receptor (PXR)) were not substantially altered. Using reporter gene assays, we show that metamizole is not acting as a direct ligand to either PXR or CAR, suggesting a phenobarbital-like mechanism of induction. These data warrant further studies to elucidate the drug-interaction potential of metamizole, especially in patients with long-term treatment.     

Structure and luminescence properties of La6Ba4(SiO4)6F2:Dy3+ phosphor with apatite structure

In this study, we investigated the structure, luminescence properties and morphology of La₆Ba₄(SiO₄)₆F₂:Dy³⁺ in detail using X-ray diffraction (XRD), photoluminescence spectroscopy, scanning electron microscopy (SEM) and decay kinetics measurements. The results indicate that La₆Ba₄(SiO₄)₆F₂:Dy³⁺ was well crystallized, and its structure is of apatite-type and belongs to the hexagonal system. The prepared samples exhibit two intense characteristic bands in the blue (484 nm) and yellow (579 nm) spectral ranges corresponding to the Dy³⁺ transitions ⁴F_9/2 → ⁶H_15/2 and ⁴F_9/2 → ⁶H_13/2, respectively. These dominant photoluminescence bands are accompanied by a weak red band (670 nm) due to the ⁴F_9/2 → ⁶H_11/2 transition. The emission color of La₆Ba₄(SiO₄)₆F₂:Dy³⁺ phosphors are found to fall in the white light region. The Dy³⁺ optimal dopant concentration in the La₆Ba₄(SiO₄)₆F₂ host was found to be 0.12 (mol). In addition, the phosphors have high thermal stability. Hence, La₆Ba₄(SiO₄)₆F₂:Dy³⁺ may have an application in white light-emitting diodes.

Single-phase white light emitting La₆Ba₄(SiO₄)₆F₂:Dy³⁺ phosphors with good thermal stability were obtained.     

Regulation of JAK3 expression in human monocytes: phosphorylation in response to interleukins 2, 4, and 7

The Janus family of kinases (JAKs) has been shown to be involved in the signal transduction of a number of cytokine receptors. Recently, we have cloned a novel JAK family member, JAK3, that is expressed in natural killer and activated T cells and is coupled functionally and physically to the interleukin 2 (IL-2) receptor in these cells. Here we report that JAK3 was expressed at low but detectable levels in human monocytes. In contrast, JAK3 expression was strongly induced during activation by interferon gamma (IFN-gamma) or lipopolysaccharide. Moreover, JAK3 became tyrosine phosphorylated in response to IL-2, IL- 4, and IL-7 but not response to IFN-gamma or granulocyte/macrophage colony-stimulating factor. Together, these findings suggest that JAK3 is functionally important in activated monocytes and cells of the myeloid lineage and is involved in signaling responses of cytokines that use the common gamma-chain of the IL-2 receptor.     

The extracellular domain of CD45 controls association with the CD4-T cell receptor complex and the response to antigen-specific stimulation

The CD45 tyrosine phosphatase plays an important role in regulating T lymphocyte activation, but the function of the different isoforms of CD45 is not known. T cell transfectants have been prepared that express individual CD45 isoforms in cells with a well-defined T cell receptor (TCR) from the D10 T helper 2 clone. We find that cells bearing low molecular weight CD45 isoforms are far more efficient in responding to stimulation with peptide and antigen-presenting cells compared with cells bearing high molecular weight CD45 isoforms. One hypothesis for the preferential activation of cells that express low molecular weight CD45 isoforms is that they interact with other cell surface antigens important in TCR signaling, altering their phosphorylation status and affecting the character of the signal transduction pathway. In this report, using cells expressing single isoforms, we demonstrate that low molecular weight isoforms of CD45 preferentially associate with CD4 and the TCR complex compared with high molecular weight isoforms. The molecular basis for this interaction was further examined using a glycosyl phosphatidyl inositol (GPI)-linked form of CD45Null (lacking tyrosine phosphatase domains), which preferentially associated with CD4 compared with GPI-linked CD45ABC, and cytoplasmic tail mutants of CD4, which retained the ability to coassociate. Using this panel of transfectants, it is clear that the interaction between CD4 and CD45 does not require the cytoplasmic domains of CD45, but is dependent on the specific external domain of the various isoforms: low molecular weight species were more likely to associate with the CD4-TCR complex than the higher molecular weight isoforms, and their ability to coassociate correlated with the magnitude of the response to specific antigen.     

Homeostatic maintenance of natural Foxp3(+) CD25(+) CD4(+) regulatory T cells by interleukin (IL)-2 and induction of autoimmune disease by IL-2 neutralization

Interleukin (IL)-2 plays a crucial role in the maintenance of natural immunologic self-tolerance. Neutralization of circulating IL-2 by anti-IL-2 monoclonal antibody for a limited period elicits autoimmune gastritis in BALB/c mice. Similar treatment of diabetes-prone nonobese diabetic mice triggers early onset of diabetes and produces a wide spectrum of T cell-mediated autoimmune diseases, including gastritis, thyroiditis, sialadenitis, and notably, severe neuropathy. Such treatment selectively reduces the number of Foxp3-expressing CD25(+) CD4(+) T cells, but not CD25(-) CD4(+) T cells, in the thymus and periphery of normal and thymectomized mice. IL-2 neutralization inhibits physiological proliferation of peripheral CD25(+) CD4(+) T cells that are presumably responding to normal self-antigens, whereas it is unable to inhibit their lymphopenia-induced homeostatic expansion in a T cell-deficient environment. In normal naive mice, CD25(low) CD4(+) nonregulatory T cells actively transcribe the IL-2 gene and secrete IL-2 protein in the physiological state. IL-2 is thus indispensable for the peripheral maintenance of natural CD25(+) CD4(+) regulatory T cells (T reg cells). The principal physiological source of IL-2 for the maintenance of T reg cells appears to be other T cells, especially CD25(low) CD4(+) activated T cells, which include self-reactive T cells. Furthermore, impairment of this negative feedback loop via IL-2 can be a cause and a predisposing factor for autoimmune disease.     

CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation

The importance of the B7/CD28/CTLA-4 molecules has been established in studies of antigen-presenting cell-derived B7 and its interaction with the T cell costimulatory molecule CD28. CTLA-4, a T cell surface glycoprotein that is related to CD28, can also interact with B7-1 and B7-2. However, less is known about the function of CTLA-4, which is expressed at highest levels after activation. We have generated an antibody to CTLA-4 to investigate the consequences of engagement of this molecule in a carefully defined system using highly purified T cells. We show here that the presence of low levels of B7-2 on freshly explanted T cells can partially inhibit T cell proliferation, and this inhibition is mediated by interactions with CTLA-4. Cross-linking of CTLA-4 together with the TCR and CD28 strongly inhibits proliferation and IL-2 secretion by T cells. Finally, results show that CD28 and CTLA- 4 deliver opposing signals that appear to be integrated by the T cell in determining the response to activation. These data strongly suggest that the outcome of T cell antigen receptor stimulation is regulated by CD28 costimulatory signals, as well as inhibitory signals derived from CTLA-4.     

The protein tyrosine kinase p56lck regulates thymocyte development independently of its interaction with CD4 and CD8 coreceptors [corrected] [published erratum appears in J Exp Med 1993 Sep 1;178(3):1135]

The lck gene encodes a lymphocyte-specific protein tyrosine kinase of the nonreceptor type that is implicated in signal transduction pathways emanating from the CD4 and CD8 coreceptors. Previous studies also support a role for p56lck in regulating T cell receptor beta gene rearrangements and, more generally, thymocyte development. Here we report that a mutant form of p56lck, which is incapable of interacting with CD4 or CD8, behaves indistinguishably from association-competent p56lck with respect to its ability to affect thymocyte maturation. The effects of p56lck remained specific in that the closely related src- family kinase p59hck was incapable of substituting for p56lck in arresting beta locus gene rearrangements. These data support the view that src-family kinases perform highly specialized and often nonoverlapping functions in hematopoietic cells, and that p56lck acts independently of its association with CD4 and CD8 to regulate thymocyte development.