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Biomedical subjects

H Wakasugi

Publications and source records attributed to H Wakasugi.

At least 109 records · Page 6Linked to original sources
PubMed1987

Epstein-Barr virus-containing B-cell line produces an interleukin 1 that it uses as a growth factor.

We report the establishment of a spontaneous interleukin 1 (IL-1)-producing subclone derived from the human Epstein-Barr virus (EBV)-containing B-lymphoblastoid cell line (721 LCL) and show that the IL-1 produced by this B-cell subclone is distinct from other types of IL-1. The parental cell line 84.5, a deletion mutant of the 721 LCL cell line, can be induced to produce IL-1 activity when stimulated by certain inducers such as phorbol 12-myristate 13-acetate in the presence of fetal calf serum. From this parental 721/84.5 clone, a subclone, termed 3B6, has been developed. This 3B6 subclone has an immature B-cell phenotype, expresses only HLA class II DP subregion antigens, and spontaneously releases IL-1 in the culture supernatant with relatively few inhibitory molecules under serum-free culture conditions. The 3B6-derived IL-1 was purified from 3B6 conditioned medium with a three-step procedure. The molecular weight of this IL-1 is 13,500, and the isoelectric point values are pH 4.9 and 5.1 without any component focusing near pH 7. The N-terminal amino acid sequence differs markedly from those reported for the two IL-1 species produced by monocytes. The purified material shares several biological properties with monocyte IL-1, since it could induce the proliferation of murine thymocytes, the production of interleukin 2 by phytohemagglutinin-stimulated cloned HSB2 T cells, and the proliferation of human fibroblasts. However, this IL-1 activity could not be blocked by polyclonal anti-monocytic IL-1 antibodies, and, more importantly, it was not pyrogenic in rabbits. Finally, it promotes the growth of B-cell clones derived from the parental 721/84.5 lines in the absence of fetal calf serum, which suggests that it could act as an autocrine growth factor in this Epstein-Barr virus-transformed B-cell line.

B-Lymphocytes↗
PubMed1986

T cell activation via CD2 [T, gp50]: the role of accessory cells in activating resting T cells via CD2.

Monoclonal antibody (MAb) GT2 defines a unique epitope on the CD2 molecule. GT2 triggers T cell mitosis in combination with any MAb directed against 9.6/T11(1) or D66, two previously defined CD2 epitopes. We have shown already that accessory cells (AC) are required for plenary T-PBL activation by any pair of Ab directed against D66 + 9.6/T11(1). In this study, we further investigated their role and found it to vary with the anti-CD2 pair used. When purified T-PBL preparation is used, the level of [3H]TdR incorporation observed with anti-(GT2 + 9.6/T11(1)) Ab was not significant; however, it did prove significant, although greatly reduced, with the other anti-CD2 pairs tested. This was due to qualitative differences in the process of T-PBL activation, and the role of AC, because: anti-(GT2 + 9.6/T11(1)) did not induce IL 2-R expression on purified T-PBL, whereas the other anti-CD2 pairs tested did; anti-(GT2 + 9.6/T11(1)) did not induce detectable IL 2 secretion from purified T-PBL, whereas the other anti-CD2 pairs tested induced a low amount; and anti-CDw18 Ab inhibited the mitogenic effect of anti-(GT2 + 9.6/T11(1)) on PBMC by preventing both IL 2-R expression and IL 2 secretion, whereas anti-CDw18 Ab enhanced the mitogenic effect of the other anti-CD2 pairs tested. Paraformaldehyde-fixed AC fully restored, and recombinant IL 1 partially restored purified T-PBL mitosis triggered by all anti-CD2 pairs tested. To induce IL 2 synthesis, the necessity to cross-link anti-CD2 Ab was demonstrated by coupling one Ab on Sepharose beads and adding the second Ab in the soluble phase: under these circumstances, anti-CD2 pairs were mitogenic solely in the presence of AC. These data can be interpreted as follows. Most anti-CD2 pairs require minimal contact between AC and T-PBL to induce plenary levels of IL 2 synthesis. When anti-(GT2 + 9.6/T11(1)) are used, additional contact is necessary, both for IL 2-R expression and IL 2 synthesis, which would include CDw18 for stabilization. We believe these differences could be related to different conformational changes on the CD2 molecule, depending on the epitope on which the antibodies bind, and could account for different signaling to T cells.

Animals↗
PubMed1986

Purification to homogeneity and NH2-terminal amino acid sequence of a novel interleukin 1 species derived from a human B cell line.

A subclone, referred to as 3B6, derived from a DR-negative EBV-transformed B cell line, has been found to spontaneously produce IL 1. 3B6-IL 1 displays a pI of 5 on FPLC chromatofocusing. It has been purified to homogeneity by a sequence of ion-exchange chromatography and affinity chromatography on procion red agarose. The homogeneous material migrated with an apparent m.w. of 13,500 on SDS-PAGE. The overall recovery of IL 1 activity was estimated at 57%. The final material had a specific activity of 7.8 X 10(6) half-maximal units/mg and represented a 50,000-fold purification. A partial NH2-terminal amino acid sequence has been obtained that is different from those reported from monocytic IL 1. However, this molecule can formally be identified as IL 1 on its spectrum of biologic activities. In addition to inducing the proliferation of murine thymocytes in the co-stimulator assay. 3B6-IL 1 is active on both human T and B cells, respectively, in inducing IL 2 synthesis by cells from a subcloned HSB 2 line and promoting the proliferation of anti-IgM-stimulated human peripheral blood B lymphocytes. Furthermore, 3B6-IL 1 acts as a growth factor for normal human fibroblasts and for the 3B6 line itself. However, 3B6-IL 1 is not pyrogenic in rabbits. Thus, the 3B6 cell line was shown to produce a new molecular species of IL 1, with respect to its NH2-terminal sequence, which shared all of the studied biologic activities of monocytic IL 1 except for pyrogenicity.

Amino Acid Sequence↗
PubMed1986

Interleukin 2 (IL 2) up-regulates its own receptor on a subset of human unprimed peripheral blood lymphocytes and triggers their proliferation.

Several reports indicate that human peripheral blood lymphocytes (PBL) seeded in culture with purified or recombinant interleukin 2 (IL 2) immediately after separation from the blood display a substantial level of proliferation at day 5 or 6, even in the absence of any activating signal. The spontaneously IL 2 proliferating cells are large lymphocytes, and they co-purify on a Percoll gradient in the large granular lymphocytes (third (LGL) fraction) together with the natural killer (NK) activity. When LGL were separated into NKH1 (an NK-specific surface marker)-positive and NKH1-negative cells by fluorescence-activated cell sorting (FACS), proliferating cells were mainly found in the NKH1-negative fraction. On the contrary, when cells from Percoll fraction 3 were separated into OKT3-negative and positive cells, the majority of the proliferating cell was found in the OKT3-positive cells. These results indicate that spontaneously IL 2 proliferating (SIP) cells most probably belong to the T cell lineage, but are distinct from NK cells. Surprisingly, cells from this Percoll fraction examined immediately after separation from the blood do not express detectable amounts of IL 2 receptors as assessed by three different techniques: binding of [3H]IL 2, binding of [125I]anti-Tac antibodies, and FACS analysis with the use of anti-Tac antibodies. However, after 18 hr of culture in IL 2-supplemented medium, 5 to 7% of these cells became Tac-positive by FACS analysis. Additional analysis of IL 2 receptor induced in culture with IL 2 was performed by [125I]anti-TAC binding and by [3H]IL 2 binding. Scatchard analysis of [3H]IL 2 binding, in the range of concentrations leading to the detection of high-affinity binding sites, showed an affinity constant similar to that of conventional phytohemagglutinin blasts. The role of IL 2/IL 2 receptor interaction in the proliferation process was confirmed by the fact that proliferation, in contrast with NK activation, was clearly inhibited by anti-Tac antibodies. When LGL activated with IL 2 for 60 hr were sorted into Tac+ and Tac- cells, equal levels of NK activity was found in the two fractions. Proliferation, however, was only observed in the Tac+ population. We interpret these results to indicate that SIP cells are preactivated cells circulating in the blood. They are large cells and represent a very small proportion of circulating lymphocytes (0.3%). They express a subliminal amount of IL 2 receptor. Cultivated in the presence of IL 2, IL 2 receptor expression is enhanced to a detectable level, and the SIP cells begin to proliferate. These SIP cells could be activated T cells present in every normal individual.

Antibodies, Monoclonal↗
PubMed1985

Accessory function of human leukemic cell lines: properties of B and B-K562 hybrid cell lines.

The accessory function (AF) of various B cell lines has been investigated by studying their ability to replace monocytes inducing the proliferation of highly purified T lymphocytes in the presence of phytohemagglutinin. AF could be exerted by B cells from different origin, including Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCL), Burkitt's lymphoma (BL)-derived lines, either EBV-positive or not, pre-B and B leukemias and lymphomas. The EBV-converted BJAB-B95 BL cells could exert an AF as efficient as BJAB cells, their EBV-negative counterparts, which demonstrates that EBV itself does not play any role in this function. The HLA-DR antigen-negative BL-K562 hybrids PUTKO and DUTKO, T51.4.1.6 cells which derived from an HLA-DR-negative variant of the T51 LCL and 721.84.5 cells from a mutagenically dissected HLA-DR-negative clone of the 721 LCL, also exerted very efficient AF. 721 LCL, PUTKO and DUTKO hybrids could produce interleukin 1 activity when triggered with 12-O-tetradecanoyl-phorbol 13-acetate (TPA). Furthermore, addition of anti-DR monoclonal antibodies which blocked completely the AF of the B cell lines in mixed lymphocyte-tumor cell reaction did not affect the mitogen-triggered AF exerted by the same B cell lines. The induction of cellular differentiation with TPA markedly reduced the AF exerted by all B cell lines studied as well as by the hybrid cells DUTKO. By contrast, the PUTKO hybrid was unsensitive to sodium butyrate and TPA treatments, which were unable to abrogate its AF. Taken together, these data indicate that the AF in mitogen-induced T lymphocyte responses is dependent upon some precise maturational stages of accessory cells and HLA-DR antigen expression is not required for AF in mitogen-induced proliferation assays.

Animals↗
PubMed1985

IL 2 receptor induction on human T lymphocytes: role for IL 2 and monocytes.

In this report we studied the requirements for the activation and proliferation of highly purified human T lymphocytes. Purified T cells incubated for 3 days with PHA neither proliferate nor express IL 2 receptors as detected by FACS analysis with the use of anti-Tac antibodies. However, purified T cells incubated with Con A or anti-T3 moAb do not proliferate, albeit 30 to 35% T cells express Tac epitopes. The addition of IL 2, either natural purified or recombinant, resulted in both the appearance of Tac antigen and the proliferation of PHA-activated T cells. Much to our surprise, IL 2 did not induce proliferation of Tac-positive T cells activated by Con A or soluble anti-T3 unless monocytes were added to the cultures. These data suggested that two classes of IL 2 receptors might exist on T cells, one of which was not functionally involved in T cell proliferation. In keeping with this interpretation, we have been able to demonstrate, using a radiolabeled IL 2 binding assay, that anti-T3 moAb induced almost exclusively IL 2 receptors of low affinity (Kd = 30 to 70 X 10(-9) M) and that additional signals, provided by monocytes, are required for the acquisition of high affinity receptors. IL 2 itself can induce high affinity receptors on PHA-stimulated T cells but not on cells activated by Con A or anti-T3. In this latter case the physical presence of monocytes is required and cannot be substituted by IL 1, thus indicating a previously unreported role for monocytes. It is postulated that the contact of monocytes with T cells induces a switch from an inactive low affinity conformation of the IL 2 receptor to a functional high affinity one.

Adult↗
PubMed1985

HLA class-I and class-II antigen expression by human leukemic K562 cells and by Burkitt-K562 hybrids: modulation by differentiation inducers and interferon.

K562 cells, which could be regarded as pluripotent hematopoietic progenitors, are usually considered as HLA class-I and class-II-negative cells. We show here that differentiation induction (with either sodium butyrate, 12-O-tetradecanoyl-phorbol-13-acetate, or teleocidin) or recombinant alpha- or gamma-interferon (IFN) treatment resulted in the augmentation of HLA class-I antigen expression. This augmentation of HLA class-I antigens was also observed in the Burkitt X K562 hybrid cells PUTKO and DUTKO (the latter coming from two presumably HLA-A, B-negative parents). HLA class-I genes are thus functional in K562 cells. In this system, alpha- and gamma-IFN had no clear differentiating capacity, since they were not able to modulate the expression of various hematopoietic markers, as chemical differentiation inducers did. On the other hand, neither differentiation induction nor interferon treatment could induce HLA class-II antigen expression on K562 cells. These molecules could be very faintly induced in PUTKO and DUTKO hybrids, in contrast with strong HLA class-II expression on the B parental lines. Whether these results are due to "lineage infidelity" in K562 cells or whether K562 cells represent the proliferation of HLA class-I-positive class-II-negative hematopoietic cells, with active suppression of HLA class-II antigen expression, is discussed.

Burkitt Lymphoma↗
PubMed1984

Effect of vitamin E on platelet aggregation in diabetes mellitus.

Vitamin E is known to be an inhibitor of platelet prostaglandin production and aggregation. The rate of platelet aggregation induced by adenosine diphosphate was significantly increased in diabetics with proliferative retinopathy and the enhanced production of thromboxane B2, a stable metabolite of thromboxane A2, was demonstrated in those patients. On the other hand, vitamin E in platelets was significantly reduced in diabetics compared with age matched controls. In addition, it was shown that vitamin E content in platelets examined in diabetic and control subjects inversely correlated with both the rate of platelet aggregation and thromboxane B2 production during aggregation. It is suggested that the reduced vitamin E levels in diabetic platelets can contribute to the mechanisms of the enhanced platelet thromboxane production and aggregation which relate to the development of vascular complications.

Blood Platelets↗
PubMed1984

K562 cells induced to differentiate by phorbol ester tumor promotors resist NK lysis.

The effect of various phorbols and phorbol diesters on the NK sensitivity of the human leukemic K562 cells was studied. A marked decrease in K562 cell susceptibility was achieved by culture in the presence of either 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or beta-phorbol-dibutyrate. The maximum protection against NK lysis was achieved when K562 cells were cultured in the presence of 160 nM TPA for 48 hr (mean percentage inhibition: 61% of specific lysis). As for untreated targets, the residual killing of K562 cells after TPA treatment was mediated through large granular lymphocytes (LGL). The experimental procedures required to achieve maximal NK protection with TPA resulted simultaneously in marked phenotypical changes in K562 cells: erythroid and early myeloid markers decreased, whereas the expression of megakaryocytic markers was increased as shown by staining with antiplatelet monoclonal antibodies and assessment of platelet peroxidase activity. Chemical phorbol analogs which were unable to induce K562 cell differentiation did not affect K562 cell sensitivity to NK lysis. De novo protein synthesis is involved in the TPA-induced NK resistance, since this effect was abolished by pretreatment of K562 cells with actinomycin D or cycloheximide. TPA has been previously demonstrated to reduce NK effector activity. In our data however, the observed TPA effects were not due to release of TPA acting on effector cells during the NK assay since TPA-treated K562 cell supernatants were unable to inhibit NK activity in control assays. Thus, TPA appears to decrease NK killing of malignant cells, both by depressing NK effector cells functions and by reducing the susceptibility to NK lysis of the target cells. In single-cell agarose assays, TPA-treated K562 cells demonstrated reduced NK-binding capacity and reduced sensitivity to lysis after binding. These defects could not be reversed by activation of the NK effector cells with interferon. The results here reported extend the previously suggested relations between the expression of NK-target structures and the differentiation stage of malignant cells.

Cell Differentiation↗
PubMed1984

Interleukin 1 can replace monocytes for the specific human B-cell response to a particulate antigen.

It is shown that the anti-trinitrophenyl (TNP) response of human B cells to trinitrophenyl polyacrylamide beads (TNP-PAA) is monocyte dependent. This response is abolished by extensive adherent cell depletion and restored by the addition of monocytes. The optimal response is obtained with 3% monocytes, higher numbers being suppressive. Supernatants from muramyl dipeptide (MDP)-activated monocytes can restore the response of monocyte-depleted preparations even when cells are cultured at suboptimal concentration. A partially purified preparation of interleukin (IL-1) has a comparable restorative ability. The following arguments suggest that monocytes do not function as antigen-presenting cells for this particulate antigen: (i) antigen-pulsed monocytes induce neither an anti-TNP response nor a specific T-cell proliferative response; (ii) allogeneic monocytes function as well as autologous monocytes to restore the response of nonadherent cells; (iii) HLA-DR-negative cells from the human leukemia cell line K562 can replace monocytes for this response. Monocyte supernatants do not replace T cells for the response of B-enriched lymphocytes, showing that T cells are directly involved in B-cell activation.

Acrylic Resins↗
PubMed1984

Effect of vitamin E on platelet aggregation in diabetes mellitus.

Vitamin E is known to be an inhibitor of platelet prostaglandin production and aggregation. The rate of platelet aggregation induced by adenosine diphosphate was significantly increased in diabetics with proliferative retinopathy, and the enhanced production of thromboxane B2, a stable metabolite of thromboxane A2, was demonstrated in those patients. On the other hand, vitamin E was significantly reduced in platelets obtained from diabetics as compared to age matched control subjects. In addition, it was shown that vitamin E content in platelets examined in diabetic and control subjects inversely correlated with both the rate of platelet aggregation and thromboxane B2 production during aggregation. It is suggested that the reduced vitamin E levels in diabetic platelets can contribute to the mechanisms of the enhanced platelet thromboxane A2 production and aggregation which lead to the development of vascular complications.

Adenosine Diphosphate↗
PubMed1984

Accessory function and interleukin 1 production by human leukemic cell lines.

Highly purified T lymphocytes do not proliferate in response to mitogens, unless adherent HLA-DR-positive monocytes are added to the culture. This accessory function (AF) of monocytes requires the release of interleukin 1 (IL 1). Cells from three human leukemic cell lines, K562, HL60, and U937, could very efficiently replace monocytes in a 72-hr mitogen-induced T cell proliferation assay. The AF was clearly related to precise maturational stages of these cells; the hematopoietic precursor K562 cells spontaneously exerted high AF, but lost this property when treated with differentiation inducers. On the contrary, the promyelocytic HL60 cells and the "histiocytic" U937 cells exhibited no spontaneous AF, but acquired this property when induced to differentiate along the granulocytic and/or monocytic pathway. Three leukemic cells could not only stimulate T cells to proliferate and produce IL 2 in the presence of mitogens, but also under appropriate culture conditions these cells could produce IL 1, which could not be distinguished from normal human monocyte derived IL 1 by gel filtration and isoelectric focusing. Moreover, analysis of phenotypic markers revealed that AF and production of IL 1 could be demonstrated in different cell types and therefore are not restricted to the monocytic lineage. No HLA-DR antigen could be detected on K562 and HL60 cells. Thus, the expression of the DR antigens is not required for AF and IL 1 production in response to mitogens. These three human leukemic cell lines will provide convenient sources of human IL 1.

Cell Line↗
PubMed1983

Comparison of somatostatin distribution in pancreatic duct ligated rats and streptozotocin diabetic rats.

The somatostatin content in the pancreas and gastrointestinal tract was examined in normal, streptozotocin (STZ)-diabetic and pancreatic duct ligation (PDL) rats. The somatostatin content in the pancreas and gastric antrum in the STZ-diabetic group showed a higher concentration compared with those in the normal control group (p less than 0.001). The somatostatin content in the duodenum in the PDL group showed a higher concentration compared with those in the sham-operated group (p less than 0.02). There was no significant increase in the lower gastrointestinal tract in the normal, STZ and PDL groups. These facts suggest that the concentration of somatostatin varies according to tissues and organs and that the distribution of somatostatin in chronic pancreatic injury caused by PDL is different from that in diabetes mellitus cases or controls.

Animals↗