The generation and regulation of lymphocyte populations: evidence from differentiative induction systems in vitro.

Results with a dual assay, for the induction of Thy-1+ T cells and of CR+ B cells from marker-negative precursors, confirm that thymopoietin is at present the only known selective inducer of prothymocytes. In contrast, various inducers, including ubiquitin, are active in both assays. Pharmacological evidence indicates that there are different cellular receptors for ubiquitin and thymopoietin. Prothymocytes and pro-CR+ B cells compose two distinct populations in bone marrow and spleen; their distribution in density gradients is different, and elimination of either population enriches the other proportionately. There are no noteworthy differences between induction of these two populations in regard to (a) kinetics, (b) dependence on temperature and protein synthesis, (c) activation by cAMP, and (d) inhibition by cGMP. The opposite inductive effects of cAMP and cGMP were corroborated by the use of pharmacological agents that raise or lower the levels of intracellular cyclic nucleotides. In contrast, a third induction assay, which monitors acquisition of the PC+ surface phenotype, indicates that this differentiative step, the last known for B cells, is initiated by cGMP and inhibited by cAMP. Induction of PC is also inhibited by thymopoietin, signifying that the inductive selectivity of thymopoietin is not due to restriction of its receptors to the T lineage cells. Rather it seems that receptors for thymopoietin occur also on PC-inducible and other B cells, although in this case geared biochemically to inhibition rather than expression of the succeeding gene program. This suggests a role for thymopoietin in the coordinated interregulation of lymphocyte classes, in addition to its better-known function as the thymic inducer of prothymocytes. Present data conform to a general scheme in which the cyclic nucleotides cAMP and cGMP, and agents that affect intracellular levels of these mediators, influence reciprocally the early and late (functional) phases of lymphocyte differentiation as a whole, while thymopoietin influences reciprocally the differentiation of the B and T classes of lymphocyte.

Disproportion in T-cell subpopulations in immunodeficient mutant hr/hr mice.

Mice of the HRS strain, which carry the mutant gene hr, were examined for abnormalities in representation of the three T-cell sets Ly1, Ly23, and Ly123 in the spleen. The salient feature of hr/hr mice, which are immunologically deficient, in comparison with +/hr segregants, was a gross disproportion in numbers of cells belonging to the Ly1 and Ly123 sets, at the age of 3--3.5 mo. At this age, Ly123 cells of hr/hr spleen outnumbered Ly1 cells by 2:1, whereas in +/hr spleens Ly123 cells were outnumbered by approximately 1:2. Cells from pooled lymph nodes of hr/hr mice did not show a correspondingly gross disporprotion of Ly1 and Ly123 cells. Total counts of splenic T cells, and of B cells, were not significantly different in hr/hr and +/hr mice.

T200 cell surface glycoprotein of the mouse. Polymorphism defined by the Ly-5 system of alloantigens.

The cell line BW5147, and a mutant T200-negative cell line derived from BW5147, were studed by immunoprecipitation and peptide mapping, with xenogeneic monoclonal anti-T200 serum and with Ly-5 alloantiserum. It appears that the Ly-5 system defines a structural polymorphism of the cell surface glycoprotein T200, and that the monoclonal anti-T200 serum defines a feature of T200 that is common to mice of both Ly-5a and Ly-5b genotypes and may be invariable in the species.

Immunological studies of mouse decidual cells. I. Membrane markers of decidual cells in the days after implantation.

Mouse decidual cell suspensions from day 6 to day 8 of gestation were prepared by enzymatic treatment with collagenase and trypsin and tested for various membrane markers. (a) Besides H-2 antigens, Thy-1 antigens are present on about 50% of the cells; this may reflect the fibroblastic origin of decidual cells or be a marker expressed on some decidual cells possibly under hormonal control. (b) T or B lymphocytes, as defined by four Lyt antigens or surface immunoglobulins, are not present in significant amounts. (c) A substantial number of cells bearing receptors for the Fc portion of IgG (FcR) is detectable in the decidua, probably closely connected with trophoblast cells; these FcR-bearing cells may act in preventing excessive invasion of uterine tissue by trophoblast or could contribute to the protection of the embryo by interacting with maternal blocking antibodies and trophoblast. No receptors for for complement were detected, even after 16-20 h in culture after trypsin treatment.

A synthetic pentapeptide with biological activity characteristic of the thymic hormone thymopoietin.

The pentapeptide arginyl-lysyl-aspartyl-valyl-tyrosine, corresponding to amino acid residues 32--36 in thymopoietin, was synthesized. In vitro, this pentapeptide induced the differentiation of murine prothymocytes to thymocytes and inhibited differentiative induction of cells of the B lineage. This combination of actions is presently unique to the parent molecule thymopoietin. In vivo, the pentapeptide reduced the high numbers of autologous rosette-forming cells normally present in the spleens of athymic mice; this also is a property of thymopoietin. These results suggest that this readily synthesized pentapeptide corresponds to an active site of thymopoietin and might serve as a therapeutic substitute for thymopoietin.

Protein kinases: six degrees of separation?

Recent evidence for cross-talk between protein kinase B (PKB) and the Raf-1 and NF-kappaB signalling pathways has provided new hints to the complex roles that PKB may play in regulating gene transcription and also raised questions about where and when these targets are relevant.

Downstream signalling events regulated by phosphatidylinositol 3-kinase activity.

The phosphatidylinositol (PI) 3-kinase family of enzymes is now known to be regulated by several different upstream pathways in response to virtually all growth factors and cytokines. In the past few years, the phosphoinositides phosphorylated at the 3-OH position of the inositol ring have been shown to be lipid second messengers that may directly or indirectly regulate the activity of several different serine/threonine kinases. Consistent with the many different cellular events in which PI 3-kinase plays an important role, a diverse group of serine/threonine kinases are regulated downstream of PI 3-kinases, including protein kinase C (PKC) isoforms, p70 S6 kinase, and PKB/Akt. This review summarises studies done primarily in the past few years that have begun to unravel these targets of PI 3-kinase activity.

Action of complement in the lysis of mouse sarcoma cells sensitized with H-2 alloantibody.

A modified cytotoxicity assay was adapted from classical erythrocytolytic assays, in which complement components were added in sequence to antibody-sensitized cells. This assay was applied to a model system in which mouse sarcoma cells were sensitized with H-2 alloantibody. The stepwise presentation of complement components combined with the stabilization of C2 by iodine treatment considerably augmented the lytic efficiency of human complement. More generally, the techniques adopted for this study provide a new model for obtaining basic information about selective reaction steps concerned in lysis of nucleated cells by alloantibody and complement. Comparisons of the lysis of sheep erythrocytes by xenoantibody with the lysis of mouse sarcoma cells by H-2 alloantibody, in our assay system with oxidized or untreated human complement, disclosed a difference in the kinetics of C142 formation, manifest in a lag phase and a protracted tmax for the sarcoma cells. These data may suggest that multiple complement-mediated functional lesions are necessary for immune lysis of nucleated cells.

Regulatory influence of thymopentin on splenic T cell sets of thymectomized and aged mice.

Thymectomy of mice aged 6-8 weeks causes a disproportion of splenic T cell sets, the Ly123 set being relatively decreased and the Ly23 set relatively increased (Ly123 decrease: Ly23 increase). A similar disproportion of splenic T cell sets was found to occur spontaneously with advancing age (12-18 months). By PA-SRBC assay, the total number of splenic Lyt+ cells is not appreciably reduced by thymectomy or by aging, but the Thy-1+ cell count falls by about 40% according to both PA-SRBC and cytotoxicity assays. Thus there is an increase in the number of Lyt+ cells expressing sub-threshold amounts of Thy-1. The following observations show that thymopentin (TP-5), a synthetic pentapeptide analogue of thymopoietin, counteracts these changes in thymectomized and aged mice. As reported previously, the capacity of C3H/HeJ female mice to reject C3H/HeJ male skin was raised by thymectomy, or with age, and treatment with TP-5 substantially normalized the rejection response. Here we correlate these findings with changes in the profile of splenic T cell sets. The splenic T cell set profile of thymectomized B6-Tlaa male and female mice was essentially restored by TP-5. The Ly123 decrease: Ly123 increase change caused by thymectomy was not associated with obviously altered proportions of Qa-1+ and Qa-1- subsets. Treatment of aged mice with TP-5 also prevented the onset of changes in splenic T cell sets that occur spontaneously with age. Thus thymectomy and aging give rise to disproportions of splenic T cell sets, and in C3H female mice to a heightened capacity for male skin rejection, both effects being largely abrogated by the TP-5 derivative of thymopoietin.

Immunological studies of mouse decidual cells. II. Studies of cells in artificially induced decidua.

Cells from artificially induced decidual tissue (deciduoma) in the mouse were examined for Thy-1 surface antigen and receptors for the Fc portion of immunoglobulin G (FcR) and compared with cells of the normal decidua from 6 to day 9 of pregnancy. It was shown that (1) Thy-1 antigen is present on the same proportion of cells in decidua and deciduoma on day 6 and day 7, (2) FcR-bearing cells can be detected in similar numbers on day 6 and day 7 but this does not increase on day 8 in deciduoma as it does in decidua, and (3) progesterone treatment after induction of decidualization allowed further increase of FcR-bearing cells in deciduoma. These results present further evidence of the similarity between deciduoma and decidua in the mouse. They indicate that these two membrane markers are present in the early decidua, regardless of the presence of an embryo, and suggest that progesterone may play a part in the increase of FcR-bearing cells in the decidua during pregnancy.

The antineoplastic ether lipid, s-phosphonate, selectively induces apoptosis in human leukemic cells and exhibits antiangiogenic and apoptotic activity on the chorioallantoic membrane of the chick embryo.

INTRODUCTION: We investigated the cytotoxic and antiangiogenic activity of the ether lipid, 2'-(trimethylammonio)ethyl 4-(hexadecyloxy)-3(S)-methoxybutane-phosphonate (termed s-phosphonate). METHOD: Cytotoxicity was determined using an XTT bioassay. Apoptosis was measured by either DNA fragmentation or immunolabelling techniques. Angiogenesis was measured using the in vivo chorioallantoic membrane (CAM) of the chick embryo. RESULTS: S-phosphonate was selectively cytotoxic towards the human leukemic cell lines, HL-60 and AML-14, whereas leukemic K-562 cells and the murine mast cell line, MC-9, were resistant to this agent at concentrations as high as 50 microM. This selectivity resulted from the induction of apoptosis (or programmed cell death) by s-phosphonate in HL-60 and AML-14 cells but not in resistant K-562 or MC-9 cells. S-phosphonate induced localized antiangiogenic effects and membrane thinning in the CAM. This concentration-dependent antiangiogenic effect was associated with apoptosis in the CAM as measured by DNA fragmentation in extracted CAM tissue. The localized areas of membrane thinning and antiangiogenesis on the CAM caused by s-phosphonate were also the only areas of the membrane in which apoptosis occurred. CONCLUSION: We conclude that s-phosphonate selectively induces apoptosis in human leukemic cells and exhibits antiangiogenic and apoptotic activity on the CAM.

The nuclear localization of 3'-phosphoinositide-dependent kinase-1 is dependent on its association with the protein tyrosine phosphatase SHP-1.

3'-Phosphoinositide-dependent protein kinase-1 (PDK1), the direct upstream kinase of Akt, can localize to the nucleus during specific signalling events. The mechanism used for its import into the nucleus, however, remains unresolved as it lacks a canonical nuclear localization signal (NLS). Expression of activated Src kinase in C6 glioblastoma cells promotes the association of tyrosylphosphorylated PDK1 with the NLS-containing tyrosine phosphatase SHP-1 as well as the nuclear localization of both proteins. A constitutive nucleo-cytoplasmic SHP-1:PDK1 shuttling complex is supported by several lines of evidence including (i) the distribution of both proteins to similar subcellular compartments following manipulation of the nuclear pore complex, (ii) the nuclear retention of SHP-1 upon overexpression of a PDK1 protein bearing a disrupted nuclear export signal (NES), and (iii) the exclusion of PDK1 from the nucleus upon overexpression of SHP-1 lacking the NLS or following siRNA-mediated knock-down of SHP-1. The latter case results in a perinuclear distribution of PDK1 that corresponds with the distribution of PIP3 (phosphatidylinositol 3,4,5-triphosphate), while a PDK1 protein bearing a mutated PH domain that abrogates PIP3-binding is excluded from the nucleus. Our data suggest that the SHP-1:PDK1 complex is recruited to the nuclear membrane by binding to perinuclear PIP3, whereupon SHP-1 (and its NLS) facilitates active import. Export from the nucleus relies on PDK1 (and its NES). The intact complex contributes to Src kinase-induced, Akt-sensitive podial formation in C6 cells.

Dissociation of cytokine-induced phosphorylation of Bad and activation of PKB/akt: involvement of MEK upstream of Bad phosphorylation.

The phosphatidylinositol 3-kinase (PI3K)-signaling pathway has emerged as an important component of cytokine-mediated survival of hemopoietic cells. Recently, the protein kinase PKB/akt (referred to here as PKB) has been identified as a downstream target of PI3K necessary for survival. PKB has also been implicated in the phosphorylation of Bad, potentially linking the survival effects of cytokines with the Bcl-2 family. We have shown that granulocyte/macrophage colony-stimulating factor (GM-CSF) maintains survival in the absence of PI3K activity, and we now show that when PKB activation is also completely blocked, GM-CSF is still able to stimulate phosphorylation of Bad. Interleukin 3 (IL-3), on the other hand, requires PI3K for survival, and blocking PI3K partially inhibited Bad phosphorylation. IL-4, unique among the cytokines in that it lacks the ability to activate the p21ras-mitogen-activated protein kinase (MAPK) cascade, was found to activate PKB and promote cell survival, but it did not stimulate Bad phosphorylation. Finally, although our data suggest that the MAPK pathway is not required for inhibition of apoptosis, we provide evidence that phosphorylation of Bad may be occurring via a MAPK/ERK kinase (MEK)-dependent pathway. Together, these results demonstrate that although PI3K may contribute to phosphorylation of Bad in some instances, there is at least one other PI3K-independent pathway involved, possibly via activation of MEK. Our data also suggest that although phosphorylation of Bad may be one means by which cytokines can inhibit apoptosis, it may be neither sufficient nor necessary for the survival effect.

Phosphatidylinositol 3-OH kinase activity is not required for activation of mitogen-activated protein kinase by cytokines.

Hemopoietic cells respond to cytokines by initiating tyrosine phosphorylation of receptors and receptor-associated proteins, leading to the activation of numerous cytosolic and membrane associated enzymes, including phosphatidylinositol 3-OH kinase (PI 3-kinase). Recent reports have suggested that PI 3-kinase may serve as an upstream activator of mitogen-activated protein (MAP) kinase. After stimulation with interleukin-3 and granulocyte-macrophage colony-stimulating factor, we show here that inhibition of MAP kinase activity by two inhibitors of PI 3-kinase, wortmannin and LY-294002, does not correlate with their ability to inhibit PI 3-kinase or p70 S6 kinase phosphorylation. Complete inhibition of phosphatidylinositol 3,4,5-trisphosphate production occurred at approximately 100 nM WM or 25 microM LY-294002, but at these concentrations, WM significantly inhibited MAP kinase activation, while LY-294002 had virtually no effect on MAP kinase activity. Furthermore, WM does not inhibit phorbol ester-mediated MAP kinase activation, but LY-294002 does. Together these results suggest WM and LY-294002 are differentially inhibiting enzymes other than PI 3-kinase that function upstream of MAP kinase.

PKB/AKT: functional insights from genetic models.

Since its discovery 10 years ago, the potential functions of protein kinase B (PKB)/AKT have been catalogued with increasing efficiency. The physiological relevance of some of the proposed mechanisms by which PKB/AKT mediates many of its effects has been questioned, and recent work using new reagents and approaches has revealed some cracks in our understanding of this important molecule, and also hinted that these effects may involve other players.