Proliferation Of Lymphoblasts Research Articles

Alteration of purine metabolism by AICA-riboside in human B lymphoblasts

The effect of 5-amino-4-imidazole-carboximide (AICA)-riboside on different pathways of purine metabolism (biosynthesis de novo, salvage pathways, adenosine metabolism, ATP catabolism) was studied in human B lymphoblasts (WI-L2). AICA-riboside markedly decreased intracellular levels of 5-phosphoribosyl-1-pyrophosphate and in consequence affected purine biosynthesis de novo and purine salvage pathways. AICA-riboside inhibited incorporation of glycine into purine nucleotides, but when formate was used as the precursor of purine biosynthesis de novo, a biphasic effect was observed. The incorporation of formate into purine nucleotides was increased by AICA-riboside at concentrations up to 2 m m but decreased at higher concentrations. Salvage of the purine bases adenine, hypoxanthine, and guanine was markedly inhibited and utilization of extracellular adenosine in B lymphoblasts was reduced by AICA-riboside. AICA-riboside increased ribose 1-phosphate concentrations and increased degradation of prelabeled ATP. No effect on the intracellular levels of orthophosphate was found. Proliferation of WI-L2 lymphoblasts was only slightly affected at concentrations of AICA-riboside below 500 μ m but markedly inhibited by higher concentrations.

Purification to homogeneity and partial characterization of cytotoxic lymphocyte maturation factor from human B-lymphoblastoid cells.

A cytokine that can synergize with interleukin 2 to activate cytotoxic lymphocytes was purified to homogeneity. The protein, provisionally called cytotoxic lymphocyte maturation factor (CLMF), was isolated from a human B-lymphoblastoid cell line that was induced to secrete lymphokines by culture with phorbol ester and calcium ionophore. The purification method, utilizing classical and high-performance liquid chromatographic techniques, yielded protein with a specific activity of 8.5 x 10(7) units/mg in a T-cell growth factor assay. Analysis of the purified protein by sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated that CLMF is a 75-kDa heterodimer composed of disulfide-bonded 40-kDa and 35-kDa subunits. Determination of the N-terminal amino acid sequences of the two subunits revealed that both subunits are not related to any previously identified cytokine. Purified CLMF stimulated the proliferation of human phytohemagglutinin-activated lymphoblasts by itself and exerted additive effects when used in combination with suboptimal amounts of interleukin 2. Furthermore, the purified protein was shown to synergize with low concentrations of interleukin 2 in causing the induction of lymphokine-activated killer cells.

Bovine interleukin 2: Biochemical and biological characterization

Interleukin 2 (IL-2), secreted by bovine peripheral blood mononuclear cells (PBL) on stimulation with concanavalin A (Con A), was purified and characterized by different chromatographic and electrophoretic techniques. The ability of IL-2 to support proliferation of Con A-stimulated bovine lymphoblasts was used to assay and quantitate IL-2 activity. Bovine IL-2 having an apparent MW of 27 000 eluted from a gel-filtration column; from an anion exchange column peak activity was detected at 190 m M NaCl. Binding of bovine IL-2 to phenyl-Sepharose gel and elution with 35–60% ethanediol indicated its hydrophobic nature. Studies on cross-species reactivity revealed that both buffalo and goat lymphocytes respond to cattle IL-2 and detected 35% of activity from a standard cattle IL-2 preparation. Sheep lymphocyte response to cattle IL-2 was negligible.

Promotion of human T lymphocyte proliferation by IL-4.

The capacity of human rIL-4 to support the proliferation of mitogen-stimulated T cells directly as well as by increasing IL-2 production or enhancing IL-2 responsiveness was investigated. IL-4 augmented proliferation of T cells stimulated with PHA, Con A, immobilized mAb to the CD3 molecular complex (OKT3), or PMA. IL-4 increased the number of mitogen-stimulated cells entering the cell cycle as well as enhancing ongoing proliferation of mitogen-activated lymphoblasts. Facilitation of initial activation by IL-4 was not inhibited by mAb to the p55 component of the IL-2R, anti-Tac, and, therefore, was not dependent on endogenous IL-2 activity. However, IL-4-mediated enhancement of ongoing T cell proliferation stimulated by PHA or OKT3 was partially but not completely blocked by anti-Tac. Analysis of the supernatants from PHA-stimulated T cell cultures indicated that IL-4 increased the production of IL-2 by mitogen-activated cells. Moreover, IL-4 increased the amount of IL-2 mRNA that accumulated in mitogen-stimulated T cells. In addition, IL-4 markedly augmented IL-2R expression by PHA-stimulated T cells. Although IL-4 promoted ongoing DNA synthesis of mitogen-stimulated T cells in an IL-2-dependent manner, it was also able to sustain their proliferation directly. Thus, IL-4 supported proliferation of PMA-activated T cells in a manner that was not inhibited by anti-Tac. Furthermore, IL-4 could augment proliferation and IL-2R expression of T cells stimulated with PHA in the presence of cyclosporin A, which blocks endogenous cytokine production or anti-Tac. Finally, IL-4 was noted to enhance proliferation of both CD4+ and CD8+ T cell subsets. The results indicate that IL-4 enhances proliferation of mitogen-activated human T cells by a number of mechanisms, including the direct promotion of cell cycle entry and subsequent DNA synthesis, enhanced production of IL-2, and increased responsiveness to IL-2 in part by up-regulation of IL-2R expression.

Inhibition of murine lymphocyte proliferation by the B subunit of cholera toxin.

Cholera toxin is known to inhibit lymphocyte activation in vitro, an effect attributed to its ability to activate adenylate cyclase and increase intracellular cyclic adenosine monophosphate. In these studies the effects of both cholera toxin (CT) and its purified binding subunit (CT-B) on lymphocyte proliferation in vitro was examined, using a variety of cell activators. We found that both CT and CT-B inhibited mitogen- and antigen-induced T cell proliferation and anti-IgM-induced B cell proliferation in a dose-dependent manner. However, only CT-inhibited lipopolysaccharide-induced B cell proliferation. Neither CT nor CT-B inhibited antigen uptake and presentation by macrophages. The CT-B preparation used was shown not to activate lymphocyte adenylate cyclase, although CT itself was a strong activator of this enzyme. Both molecules had to bind to the lymphocyte surface in order to inhibit. The time course of inhibition of both CT and CT-B was similar in that either could be added up to 24 hr after culture initiation and still inhibit substantially. The addition of excess human recombinant interleukin 2 to the cultures did not affect the inhibition by CT, and had only a partial affect on inhibition by CT-B. Similarly, CT was able to substantially inhibit recombinant interleukin 2-dependent T lymphoblast proliferation, whereas CT-B had only a small inhibitory effect. Inhibition was not major histocompatibility complex-restricted. We conclude that the binding of CT or CT-B to the lymphocyte surface membrane interferes in some way with the activation mechanism leading to proliferation. The inhibition mediated by CT-B does not involve the stimulation of intracellular adenylate cyclase. CT appears to inhibit both by binding via its B subunit and by activation of adenylate cyclase via its A subunit.

T Cell Hyperreactivity in Obese Strain (OS) Chickens. Different Mechanisms Operative in Spleen and Peripheral Blood Lymphocyte Activation

The enhanced T cell reactivity (ConA hyperresponsiveness and IL 2 hypersecretion) of spleen lymphocytes of Obese strain (OS) chickens with spontaneous autoimmune thyroiditis has recently been shown to be due to a defect in macrophage-derived non-specific suppressor factors that regulate IL 2 secretion and IL 2-promoted T lymphoblast proliferation in normal healthy animals. In the present study, we present several lines of evidence that the increased T cell response of peripheral blood lymphocytes (PBL) of OS chickens is due to mechanisms entirely different from the described dysregulation of splenic T cells: 1) In contrast to the splenic macrophages, peripheral blood monocytes of OS animals are not deficient in the production of IL 2 antagonistic activity (IAA); 2) therefore, cocultivation of PBL from OS and Normal White Leghorn (NWL) chickens in communicating culture chambers did not abrogate the difference in Con A response as previously observed with spleen lymphocytes. 3) Immunofluorescence with a monoclonal antibody (INN CH 16) against the chicken IL 2 receptor revealed enhanced numbers of mitogen activatable T cells in OS PBL but not OS spleen lymphocytes. 4) After prolonged Con A stimulation of PBL, OS and NWL lymphoblasts did not differ from each other in functional aspects. In contrast to this, Con A lymphoblasts from OS spleens exhibited enhanced staining with INN CH 16 in parallel with an increased proliferative response to IL 2. Thus, the primary T cell dysfunction involved in the development of autoimmune disease in OS chickens is the result of at least two separate regulatory defects.

Lack of mitogenic effects of growth hormone on human leukemic lymphoblasts.

To determine whether human growth hormone (HGH) can cause proliferation of human leukemic lymphoblasts, we studied colony formation in semi-solid medium of MOLT 4, a cell line derived from an adolescent with acute lymphoblastic leukemia (ALL). Although exposure to single doses of HGH in supraphysiologic concentrations resulted in almost two-fold increases in number of colonies compared with control samples, physiologic concentrations had no effect. Similarly, physiologic concentrations of HGH had no effect on thymidine incorporation in short-term cultures of fresh lymphoblasts from children with ALL. In addition, total white blood cell and differential counts in 14 children with isolated growth hormone deficiency were reviewed pre- and post-treatment with HGH. In no case was there evidence of in vivo lymphocytosis or blastogenesis.

Concanavalin A responsiveness and interleukin 2 production in the snake Spalerosophis diadema

Thymocytes and splenocytes (SC) of adult snakes, Spalerosophis diadema, responded toconcanavalin A (Con A) in vitro by strong proliferation during the spring and autumn seasons. Con A-mediated mitogenesis was, however, abrogated in summer and winter. Conditioned medium (CM) collected from snake SC cultures stimulated with Con A in spring or autumn could enhance the Con A summer and winter responses and support the proliferation of splenic lymphoblasts. Gel filtration of native CM on Sephadex G-100 revealed the presence of two biologically active peaks of molecular weight 39–42 and 15 KD. However, only one peak of activity corresponding to molecular weight (m.w.) of 14–15 KD was observed when CM was subjected to analysis by sodium dodecyl sulphate gel electrophoresis (SDS-PAGE). The active molecular forms exhibited isoelectric points of 5.5–5.8 and 6.4–6.6. The findings suggest that Con A activation of snake lymphocytes in optimal seasonal conditions is associated with the secretion of a lymphokine analogous to the interleukin 2 (IL 2) of endothermic vertebrates.

In vitro T cell hyperreactivity in Obese strain (OS) chickens is due to a defect in nonspecific suppressor mechanism(s).

Spontaneous autoimmune thyroiditis of OS chickens is associated with a marked hyperreactivity of the T cell system. The purpose of the present study was to investigate the underlying regulatory mechanisms. Co-cultivation experiments between Con A-stimulated OS and NWL lymphocytes in communicating cultures revealed soluble regulatory factors to be responsible for the observed functional differences: the high proliferative response to Con A and hyperproduction of IL 2 of OS cells was found to be due to a deficiency in the conditioned medium of dialyzable inhibitory factor(s) that regulate IL 2 secretion of NWL lymphocytes. Furthermore, sera of young NWL chickens were found to profoundly inhibit the IL 2-promoted lymphoblast proliferation. This IL 2 antagonizing activity is lost with age (3 to 6 yr) and was found to be significantly diminished in OS birds throughout ontogeny, thus pointing to possible parallels between immune regulatory dysfunction in autoimmunity and in physiologic aging. Both enhanced T cell response and the defect in serum suppressor were inherited by (OS X CB)F1 animals, indicating that these two aberrations may be related to each other.

CUTANEOUS MALIGNANT LYMPHOMAS

Clinical and histological findings in 37 cases of cutaneous lymphomas other than mycosis fungoides and Sezary syndrome were investigated; there were 31 in adults and 6 in children. Cutaneous lesions were the first manifestations of the diseases in all cases, and they appeared mostly as tumors or nodules. Cytomorphologically, about a half of the cases showed proliferations of large cleaved, non-cleaved cells or immunoblasts (Group I). Eight cases showed a polymorphous appearance containing convoluted cells of various size (Group II). Five cases in children demonstrated monomorphous proliferation of uniform-sized lymphoblasts (Group III). The cytologic findings in 6 cases did not fit into any lymphoid groups (Group IV). The clinical findings observed in each group were reviewed and compared. Follow-up study revealed that the prognosis of Group I was the poorest among the four groups.

CELLULAR INTERACTIONS IN MARROW-GRAFTED PATIENTS

Peripheral blood mononuclear leukocytes (cells of marrow donor origin) from 89 patients were collected at various times after allogeneic marrow transplantation, stimulated in vitro by phytohemagglutinin, and assayed for the production of interleukin 2 (IL-2). This was done by testing culture supernatants for their ability to induce proliferation of human lymphoblasts and/or IL-2-dependent cultured murine cytotoxic cells. Supernatants from cultures of patient cells were compared with those of marrow donor cells. Supernatants produced by cells from most short-term marrow recipients (30-101 days postgrafting), regardless of the presence or absence of acute graft-versus-host disease (GVHD), and those from most long-term patients with chronic GVHD (103-1932 days postgrafting) had significantly lower-than-normal IL-2 activity, whereas cells from most long-term marrow recipients without GVHD (353-1934 days postgrafting) had essentially normal IL-2 activity. Additionally, we tested the ability of monocytes from 35 marrow recipients to produce interleukin 1 (IL-1) in response to lipopolysaccharide as compared with monocytes from marrow donors or normal unrelated individuals. IL-1 activity in culture supernatants of patient cells, regardless of the time of testing after marrow grafting and the status of GVHD, was found not to differ from that in supernatants of normal cells. These findings suggest that impaired T cell functions seen in some (but not all) marrow recipients are probably not due to IL-1 but to IL-2 deficiency or to the mechanism that causes IL-2 deficiency.

Evidence for the existence of an IL-2 like lymphocyte growth promoting factor in a bony fish, [formula omitted][formula omitted

Activity promoting the growth of carp T-like cells has been found in supernatants of mitogen (PHA)- and alloantigen (MLR)-stimulated carp leukocyte cultures. Activity level in culture supernatants was elevated by phorbol myristate acetate. Proliferation of carp T-like lymphoblasts was also promoted in the presence of Il-2-containing supernatants of mammalian origin. The significance of these findings is discussed.

Inhibition by somatostatin of the proliferation of T-lymphocytes and Molt-4 lymphoblasts

The proliferation of Molt-4 lymphoblasts and phytohemagglutinin-stimulated human T lymphocytes in vitro was inhibited significantly by 10 −12to 10 −10 M and 10 −13to 10 −9 M somatostatin 14, as assessed by the uptake of [ 3H]thymidine and [ 3H]leucine, respectively. The inhibitory effect of somatostatin 14 was not attributable to cytotoxicity and was associated with a mean degradation of 52 and over 95% of immunoreactive somatostatin, respectively, after 3 and 24 hr of incubation at 37 °C. The specific suppression of Molt-4 lymphoblasts and T lymphocytes by somatostatin represents a distinct mechanism for the specific regulation of immunological responses by neuropeptides of the peripheral nervous system and gastrointestinal tract.

Altered thymic epithelial cells may be decisive for Moloney virus-induced lymphoma development

Infection of newborn mice with Moloney leukemia virus (M-MuLV) causes a T-cell differentiation block in the thymic cortex accompanied by proliferation and accumulation of prethymic lymphoblasts in the thymus and subsequent spreading of these cells to generate systemic lymphoma. Current evidence shows that thymic reticular epithelial cells (REC) provide a microenvironment necessary for the maturation of prethymic lymphoblasts to mature T-lymphocytes by secretion of various thymic factors. A change in that environment due to infection of REC by virus could be decisive for the failure of lymphoblasts to mature and thus contribute to lymphoma development.We have studied the morphology and distribution of the major thymic cell populations at different stages of tumorigenesis in Balb/c mice infected when newborn with 0.2ml M-MuLV suspension, 6.8 log FFU/ml. Thymic tissue taken at 1-2 weekly intervals up to tumor development was processed for light and electron microscopy, using glutaraldehyde-OsO4fixation and Epon-Araldite embedding.

Anisakis simplex and Terranova sp.: Inhibition by larval excretory-secretory products of mitogen-induced rodent lymphoblast proliferation

Execretory-secretory products were collected from supernatants of in vitro cultures of larval nematodes, Anisakis simplex (type I) and Terranova sp. (Hawaii type A). These materials were found to be more potent inhibitors of rodent lymphocyte blast transformation induced by concanavalin A and bacterial lipopolysaccaride than whole worm extracts of the same parasites. Inhibition of blast transformation was a result of cytostatic rather than toxic effects on proliferating lymphoid cells. The material(s) responsible for suppression are greater than 10,000 MW and are heat labile.

The biochemical and clinical consequences of 2'-deoxycoformycin in refractory lymphoproliferative malignancy

A deficiency of adenosine deaminase, an enzyme important in purine nucleoside catabolism, is associated with a severe combined immunodeficiency disease in children. Inhibition of this enzyme in vitro and in vivo results in an impairment in lymphoblast proliferation. We have investigated the pharmacologic inhibition of this enzyme by 2′-deoxycoformycin in 15 patients with hematologic malignancies. Biochemical consequences of the administration of this agent were closely monitored in erythrocytes, nucleated peripheral blood and bone marrow cells, serum, and urine. A marked rise in erythrocyte dATP was accompanied by a depletion of ATP in those patients exhibiting toxicity. Most patients excreted large amounts of deoxyadenosine but not adenosine in the urine. Serum deoxyadenosine rose in patients demonstrating a marked decrease in cell mass. The biochemical disturbances and clinical toxicity, including hepatic, renal, and conjunctival abnormalities, were usually reversible. Central nervous system toxicity, which potentially was the most serious consequence, was associated with high erythrocyte dATP/ATP ratios and high levels of cerebrospinal fluid deoxyadenosine. In patients with lymphoma and leukemia, objective responses were observed but were short- lived. Patients with chronic lymphocytic leukemia receiving weekly low doses of the drug demonstrated minimal toxicity and some efficacy. The chemotherapeutic potential o 2′-deoxycoformycin, as either a single agent or in combination with Ara-A, merits further exploration.

The Biochemical and Clinical Consequences of 2’-Deoxycoformycin in Refractory Lymphoproliferative Malignancy

A deficiency of adenosine deaminase, an enzyme important in purine nucleoside catabolism, is associated with a severe combined immunodeficiency disease in children. Inhibition of this enzyme in vitro and in vivo results in an impairment in lymphoblast proliferation. We have investigated the pharmacologic inhibition of this enzyme by 2'-deoxycoformycin in 15 patients with hematologic malignancies. Biochemical consequences of the administration of this agent were closely monitored in erythrocytes, nucleated peripheral blood and bone marrow cells, serum, and urine. A marked rise in erythrocyte dATP was accompanied by a depletion of ATP in those patients exhibiting toxicity. Most patients excreted large amounts of deoxyadenosine but not adenosine in the urine. Serum deoxyadenosine rose in patients demonstrating a marked decrease in cell mass. The biochemical disturbances and clinical toxicity, including hepatic, renal, and conjunctival abnormalities, were usually reversible. Central nervous system toxicity, which potentially was the most serious consequence, was associated with high erythrocyte dATP/ATP ratios and high levels of cerebrospinal fluid deoxyadenosine. In patients with lymphoma and leukemia, objective responses were observed but were short-lived. Patients with chronic lymphocytic leukemia receiving weekly low doses of the drug demonstrated minimal toxicity and some efficacy. The chemotherapeutic potential o 2'-deoxycoformycin, as either a single agent or in combination with Ara-A, merits further exploration.

Altered regulation of Epstein-Barr virus induced lymphoblast proliferation in rheumatoid arthritis lymphoid cells.

AbstractAn empiric observation of the rapid development of B lymphoblast cell lines after in vitro infection with Epstein‐Barr virus (EBV) in peripheral blood mononuclear cells (PBM) from patients with rheumatoid arthritis (RA) led us to compare cellular regulation of EBV‐induced lymphoblast outgrowth in RA and normal lymphoid cell populations. We found that RA PBM developed proliferating B lymphoblast colonies in a median time of 10 days after exposure to an EBV containing culture supernatant, while normal PBM took 18 days to grow out under the same conditions. The more rapid outgrowth of RA cells was not due to therapy with antiinflammatory or antirheumatic drugs. The time to outgrowth of the normal cells decreased to 11 days after T cell depletion. T cell depletion of the RA cells had less effect on the time to outgrowth but it was shortened to 8 days. B lymphoblast cell lines were established from “purified” T cell populations from 18 of 20 RA patients, while populations of T cells from normal donors with the same percentage of T and B cells did not produce lymphoblast cell lines when infected with EBV. In addition, we found that the non‐ T lymphoid cells from 7 of 20 RA patients established permanent B lymphoblast cell lines spontaneously without EBV infection. Spontaneous outgrowth of lymphoid cells from normal donors occurred only once in 48 experiments. These results indicate abnormalities in cell‐mediated regulation of EBV‐induced cell proliferation in lymphoid cells from patients with RA.

Immunomodulatory effects of cyclomunine in vitro

Cyclomunine, a cyclic polypeptide extracted from Fusarium equiseti, is thought to be an immuno-modulator. Low concentration of cyclomunine (1 ng/ml) decreases lymphocyte nuclear refringence and potentiates the response to suboptimal concentrations of concanavalin A (Con A) in the absence of an effect on proliferation. At this concentration, cyclomunine potentiates Con A-induced suppressor cell function. Cyclomunine at concentrations of 1–10 μg/ml increases lymphocyte nuclear refringence and inhibits the proliferative responses of human lymphocytes to mitogens and of guinea-pig macrophages to a lymphokine. The inhibitory effect of cyclomunine is observed on the proliferation of mitogen-induced lymphoblasts and of human leukemic cells at a 10-fold lower concentration than of macrophages. Cyclomunine's cytotoxic index correlates with the growth inhibitory index for leukemic cells and mitogen-induced lymphoblasts. Resting lymphocytes are more resistant to the cytotoxic effects. The data indicate that cyclomunine at low concentrations may have selective effects to modulate lymphocyte helper or suppressor function, while at higher concentrations is toxic to lymphocytes. Collectively, the results indicate that cyclomunine is a new immunoregulatory compound with potent biological activity.

Kinetics of proliferation, cell differentiation, and IgM secretion in the omental lymphoid organ of B10/Sn mice following intraperitoneal immunization with sheep erythrocytes

At different times after single and repeated intraperitoneal injections of SRBC into B10/Sn mice, the [ 3H]thymidine uptake of omental lymphoid organ (OLO) cells was examined by means of scintillation counting and autoradiographic methods. The specific immune response was assayed by the PFC technique. The first SRBC injection induced, in OLO, proliferative changes in reticulum cells and an intense infiltration with lymphocytes; however, the production of humoral anti-SRBC antibodies was absent. The second immunization provoked the proliferation of lymphoblasts, the appearance of plasmocytes, and the production of specific antibodies of the IgM class only. The results indicate that OLO, thanks to the particular ability of its reticulum cells, presents a microenvironment selectively trapping B lymphocytes and limiting their functional differentiation.