Phytohemagglutinin-activated Peripheral Blood Lymphocytes Research Articles

Differential Expression of Human Topoisomerase IIIα during the Cell Cycle Progression in HL-60 Leukemia Cells and Human Peripheral Blood Lymphocytes

Human topoisomerase IIIα (huTop IIIα) has been demonstrated to belong to type IA subfamily. In this study, we found that huTop IIIα expressed constitutively and remained at high levels throughout the cell cycle in HL-60 cells when compared to the cell-cycle-dependent expression of huTop IIIα in phytohemagglutinin-activated peripheral blood lymphocytes. During the cell cycle progression, this protein remained accentuated in the nucleolus without significant translocation from the nucleolus to the nucleoplasm. In addition, during the course of granulocytic maturation in DMSO-treated HL-60 cells, huTop IIIα levels decreased when cells stopped proliferation and nucleoli diminished in size. However, its level remained unchanged during the course of monocytic maturation of vitamin D3-treated HL-60 cells which still retained its proliferative capacity and did not change the size of the nucleolus. The data suggested that huTop IIIα is involved in rDNA metabolism, such as rDNA transcription. Its cellular level appeared to be under control during the cell cycle progression of normal lymphocytes, but was found to be deregulated in HL-60 cells which may be associated with the tumor transformed cell phenotypes.

Dual role of the actin cytoskeleton in regulating cell adhesion mediated by the integrin lymphocyte function-associated molecule-1.

Intracellular signals are required to activate the leukocyte-specific adhesion receptor lymphocyte function-associated molecule-1 (LFA-1; CD11a/CD18) to bind its ligand, intracellular adhesion molecule-1 (ICAM-1). In this study, we investigated the role of the cytoskeleton in LFA-1 activation and demonstrate that filamentous actin (F-actin) can both enhance and inhibit LFA-1-mediated adhesion, depending on the distribution of LFA-1 on the cell surface. We observed that LFA-1 is already clustered on the cell surface of interleukin-2/phytohemagglutinin-activated lymphocytes. These cells bind strongly ICAM-1 and disruption of the actin cytoskeleton inhibits adhesion. In contrast to interleukin-2/phytohemagglutinin-activated peripheral blood lymphocytes, resting lymphocytes, which display a homogenous cell surface distribution of LFA-1, respond poorly to intracellular signals to bind ICAM-1, unless the actin cytoskeleton is disrupted. On resting peripheral blood lymphocytes, uncoupling of LFA-1 from the actin cytoskeleton induces clustering of LFA-1 and this, along with induction of a high-affinity form of LFA-1, via "inside-out" signaling, results in enhanced binding to ICAM-1, which is dependent on intact intermediate filaments, microtubules, and metabolic energy. We hypothesize that linkage of LFA-1 to cytoskeletal elements prevents movement of LFA-1 over the cell surface, thus inhibiting clustering and strong ligand binding. Release from these cytoskeletal elements allows lateral movement and activation of LFA-1, resulting in ligand binding and "outside-in" signaling, that subsequently stimulates actin polymerization and stabilizes cell adhesion.

Immune complexes from HIV-1+ patients contain infectious virus able to infect normal lymphocytes

It has been suggested that circulating immune complexes containing HIV-1 could be involved in enhancement of the infection through Fc receptors. To test this hypothesis, immune complexes precipitated from the sera of 25 HIV-1–seropositive individuals, at different stages of the disease, were assayed for the presence of infectious virus. When added to phytohemagglutinin-activated peripheral blood lymphocytes, seven of 25 complexes were able to sustain a productive infection. This was demonstrated by release of HIV-p24 antigens in the supernatant of activated but not resting peripheral blood lymphocytes after several days of culture. Moreover, peripheral blood lymphocytes cultured with complexes from HIV-1+ patients but not from control subject, contained integrated HIV-1 provirus in their DNA. These results demonstrated the presence of infectious virus in immune complexes from HIV-1+ patients. (J A LLERGY C LIN I MMUNOL 1996;98:827-30.)

Identification of human granzyme B promoter regulatory elements interacting with activated T-cell-specific proteins: implication of Ikaros and CBF binding sites in promoter activation.

Granzyme B serine protease is found in the granules of activated cytotoxic T cells and in natural and lymphokine-activated killer cells. This protease plays a critical role in the rapid induction of target cell DNA fragmentation. The DNA regulatory elements that are responsible for the specificity of granzyme B gene transcription in activated T-cells reside between nt -148 and +60 (relative to the transcription start point at +1) of the human granzyme B gene promoter. This region contains binding sites for the transcription factors Ikaros, CBF, Ets, and AP-1. Mutational analysis of the human granzyme B promoter reveals that the Ikaros binding site (-143 to -114) and the AP-1/CBF binding site (-103 to -77) are essential for the activation of transcription in phytohemagglutinin-activated peripheral blood lymphocytes, whereas mutation of the Ets binding site does not affect promoter activity in these cells.

Human Renal Cell Carcinoma Cells Are Sensitive to the Cytotoxic Effect of a Chimeric Protein Composed of Human Interleukin-4 and Pseudomonas Exotoxin

We have previously demonstrated that functional high-affinity interleukin-4 receptors (IL-4R) are expressed on human renal cell carcinoma (RCC) cells (N. I. Obiri et al., J. Clin. Invest. 91, 88, 1993). In the present study, we examined the cytotoxic effect (determined by inhibition of protein synthesis) of a chimeric protein composed of human IL-4 and Pseudomonas exotoxin (PE) on human RCC tumor samples obtained from patients undergoing nephrectomy. The chimeric gene encoding hIL4-PE4E was constructed by fusing a cDNA clone for human IL-4 to the 5′ end of a mutated cDNA encoding a full-length PE molecule. This gene was expressed in Escherichia coli, and large quantities of this recombinant protein were isolated to more than 95% purity. This chimeric protein, hILA-PE4E, was highly cytotoxic to all six RCC cell lines examined. The concentration of hIL4-PE4E at which 50% inhibition of protein synthesis was obtained ranged from <1 ng/ml (12 p M) to 10 ng/ml (120 p M) in five of the six isolates of RCC and 40-70 ng/ml in one other. A mutant chimeric protein which can bind to IL-4R but lacks the ADP ribosylation activity of PE was not cytotoxic to the RCC cells. The cytotoxic effect of hILA-PE4E was IL-4R mediated because a fourfold molar excess of IL-4 abrogated the cytotoxic effect of hIL4-PE4E. A neutralizing monoclonal antibody to IL-4 also abrogated the cytotoxic effect of hILA-PE4E. hIL4-PE4E showed very little cytotoxic activity to a normal human umbilical vein endothelial cell line (ID 50 = 1000 ng/ml) and a human fibroblast cell line (ID 50 ∼ 400 ng/ml). Nonactivated human peripheral blood lymphocytes (PBL) were also insensitive to hIL4-PE4E (ID 50, ∼500 ng/ml), whereas phytohemagglutinin-activated PBL were highly susceptible to the cytotoxic effect of hIL4-PE4E (ID 50, ∼4 ng/ml). These data indicate that hIL4-PE4E may be a useful agent for the treatment of human RCC without affecting normal and resting immune cells.

A wide range of human cancers express interleukin 4 (IL4) receptors that can be targeted with chimeric toxin composed of IL4 and Pseudomonas exotoxin

A chimeric toxin has been constructed by fusion of a gene encoding human interleukin 4 (hIL4) to a gene encoding a mutant form of Pseudomonas exotoxin A (PE) which cannot bind to its receptors (PE4E). The chimeric gene was expressed in Escherichia coli where large amounts of the chimeric toxin, hIL4-PE4E, was produced. Purified hIL4-PE4E was very cytotoxic to cancer cell lines of both hematopoietic and solid tumor origin. In the HUT 102 T cell leukemia and Daudi B cell lymphoma cell lines, protein synthesis was inhibited by 50% (ID50) at a hIL4-PE4E concentration of 2 and 7 ng/ml (25 and 86 pM, respectively). hIL4-PE4E was also very cytotoxic to cell lines derived from carcinomas of the colon, breast, stomach, liver, adrenals, and prostate, as well as melanoma and epidermoid carcinoma, indicating that hIL4 receptors are widely expressed on human malignancies. We also found that human phytohemagglutinin-activated peripheral blood lymphocytes were extremely sensitive to hIL4-PE4E with an ID50 of 0.2 ng/ml (2.5 pM). The cytotoxic action of hIL4-PE4E was specific because it was blocked by an excess of hIL4 and not of human interleukin 2. In addition, hIL4-PE4ED553, an enzymatically inactive form of the chimeric toxin, was not cytotoxic. These results suggest that the hIL4 receptor may be a target for therapy in malignant and immunologic disorders using hIL4 chimeric toxin.

Induction of CD8 antigen and suppressor activity by glucocorticoids in a CEM human leukemic cell clone

The relationship between glucocorticoid effect and regulation of cell surface antigens was investigated in two models of leukemic cell lines, CEM C7 denoted (r+, ly+) and CEM C1 (r+, ly−). The reactivity of murine monoclonal antibodies, anti-CD4-FITC, anti-CD8-FITC, anti-CD2-FITC and anti-calla-FITC, were analyzed using flow cytometry. The suppressor function was determined using [ 3H]thymidine incorporation into phytohemagglutinin-activated peripheral blood lymphocytes. Dexamethasone treatment of a human leukemic cell clone CEM C7 caused an increase in a subset of cells expressing the surface antigen CD8, which is present on suppressor and cytotoxic T-lymphocytes. By comparison, there was no modification of the expression of CD4 antigen, which is expressed at high levels in these cells. After two days of treatment with 5 × 10 −8 M dexamethasone, CEM C7 cells showed a two-fold increase in suppressor activity compared to untreated cells. In contrast, there was no regulation by glucocorticoids of either the CD8 or CD4 antigens in the leukemic clone CEM C1. Furthermore, no modification of the suppressor function in CEM C1 cells by dexamethasone was observed. In the human leukemic cells studied here, the ability to induce CD8 antigen expression in a CD4+ cells correlates with the ability to induce cell lysis in a glucocorticoid receptor positive cell population.

Immunological Study of Interleukin-2 in the Patients with Malignant Brain Tumors

The authors studied the capacity of peripheral blood lymphocytes from patients with malignant brain tumors to produce, and to respond to, interleukin-2 (IL-2). The role of IL-2 in generation of T cells cytotoxic to tumor cells was also studied. Lymphocytes from the patients with malignant brain tumors produced less IL-2 than did those of normal controls. However, phytohemagglutinin-activated peripheral blood lymphocytes from normal controls and the patients responded equally well to IL-2. This indicates that IL-2 receptors are abundant in the patients' lymphocytes, although IL-2 production may be depressed. Furthermore, after incubation with IL-2, lymphocytes from patients with malignant glioma exhibited higher natural killer activity and strong cytotoxicity to glioma cells. Its tumor cell cytotoxicity makes IL-2 a likely candidate for use in adoptive immunotherapy.

1 alpha,25-Dihydroxyvitamin D3 inhibits gamma-interferon synthesis by normal human peripheral blood lymphocytes.

1 alpha,25-Dihydroxyvitamin D3 [1,25-(OH)2D3], the biologically active metabolite of vitamin D3, inhibited synthesis of gamma-interferon (IFN-gamma) by phytohemagglutinin-activated peripheral blood lymphocytes (PBLs). A significant reduction of IFN-gamma protein levels in PBL culture medium was achieved with a physiologic 1,25-(OH)2D3 concentration (0.1 nM). 1,25-(OH)2D3 also inhibited accumulation of IFN-gamma mRNA in activated PBLs in a dose-dependent fashion. The ability of 1,25-(OH)2D3 to modulate IFN-gamma protein synthesis was unaltered in the presence of high concentrations of recombinant human interleukin 2. The suppression of IFN-gamma synthesis by PBLs was specific for 1,25-(OH)2D3; the potencies of other vitamin D3 metabolites were correlated with their affinities for the cellular 1,25-(OH)2D3 receptor. The time course of 1,25-(OH)2D3 receptor expression in phytohemagglutinin-activated PBLs was correlated with the time course of 1,25-(OH)2D3-mediated inhibition of IFN-gamma synthesis. In selected experiments, T-lymphocyte-enriched cell preparations were utilized. In these experiments, 1,25-(OH)2D3 was equally active as in PBL preparations. Finally, we examined the effects of 1,25-(OH)2D3 on the constitutive IFN-gamma production by two human T-lymphocyte lines transformed by human T-lymphotropic virus type I. The cell lines were established from a normal donor (cell line S-LB1) and from a patient with vitamin D-dependent rickets type 2 (cell line Ab-VDR). IFN-gamma synthesis by S-LB1 cells was inhibited in a dose-dependent fashion by 1,25-(OH)2D3, whereas IFN-gamma synthesis by Ab-VDR cells was not altered by 1,25-(OH)2D3. The data presented in this study provide further evidence for a role of 1,25-(OH)2D3 in immunoregulation.

Human macrophage-activating factors for cytotoxicity. I. Establishment of a human T-cell hybridoma that produces macrophage-activating factors for cytotoxicity.

Human T cell hybridoma, H3-E9-6, that produces macrophage activating factors for cytotoxicity (MAF-C) was prepared by somatic fusion of phytohemagglutinin-activated peripheral blood lymphocytes with emetine/actinomycin D-treated cloned human acute lymphocytic leukemia cells (CEM 11). The activities of the following were assayed: (1) macrophage-activating factor for cytotoxicity of monocytes (MAF-C 1 day), (2) macrophage-activating factor for cytotoxicity of monocyte-derived macrophages (MAF-C 6 day), (3) macrophage-activating factor for cytotoxicity of murine macrophages (MAF-Cm), (4) macrophage-activating factor for glucose consumption (MAF-G), (5) macrophage-activating factor for O2- formation (MAF-O). The culture supernatant of H3-E9-6 showed MAF-C 1 day-MAF-C 6 day, MAF-Cm, and MAF-G activities. The MAF-Cm activity was considerably enhanced by the addition of murine recombinant interferon gamma (rIFN-gamma). The MAF-C 1 day activity in the H3-E9-6 sup was not decreased by heat treatment (56 C, 30 min), by pH 2 treatment or by the addition of monoclonal anti-human IFN-gamma antibody or polymyxin B. These data suggest that MAF-C in H3-E9-6 sup is distinct from human IFN-gamma or lipopolysaccharide (LPS).

Differentiation of human T-lymphoid leukemia cells into cells that have a suppressor phenotype is induced by phorbol 12-myristate 13-acetate.

Treatment of cultured human T-lymphoid (CEM) leukemia cells with nanomolar concentrations of phorbol 12-myristate 13-acetate (PMA) resulted in a reduction in cell growth and in the acquisition of a surface antigenic pattern that is common to both suppressor and cytotoxic T lymphocytes. This antigenic pattern was detected by OKT monoclonal antibodies. PMA treatment did not cause the expression of a cytotoxic function but rather induced the expression of a suppressor cell marker. This marker was characterized by the ability of the treated CEM cells to suppress [3H]thymidine incorporation into phytohemagglutinin-activated peripheral blood lymphocytes. After 4 days of treatment of CEM cells from either cloned or the parental cell population with 16 nM PMA, 71-98% of the cells expressed reactivity with OKT3 and OKT8 antibodies whereas reactivity with OKT4 and OKT6 was detected in less than or equal to 1-8% of the cells. The CEM cells can be divided into five groups based on the antigenic patterns of cells from randomly isolated clones. The cells from four of these groups were characterized by either low or high reactivity with each of the four OKT antibodies. The antigenic pattern of the fifth group resembled that of the parent CEM cells. The acquisition of reactivity with the OKT3 antibody in the CEM cells after PMA treatment was dependent on both time and dose and did not require cell replication. Acquisition of reactivity with OKT3 antibody also occurred after treatment with phorbol 12,13-dibutyrate but not after treatment with phorbol 13-monoacetate, phorbol 12,13-diacetate, or dimethyl sulfoxide. These results indicate that treatment of CEM cells with PMA and related agents can cause the cells to express a phenotype that resembles that of a mature suppressor T lymphocyte.