Blastogenesis Of Human Lymphocytes Research Articles

The effect of calyx infusion of hibiscus sabdariffa l. On t-cells-mediated immune response in mitogen-induced blastogenesis of human lymphocytes in vitro

Free radical production and disturbance in the redox status may modulate the expression of a variety of immune and inflammatory molecules. Antioxidants could inhibit the proliferation of T lymphocytes induced by mitogens. The aim of this study was to investigate the effect of the infusion of dried calyx of Hibiscus sabdariffa L. on T-cell-mediated immune response. We examined the effect of the infusion on spontaneous and mitogen-induced blast transformation of human lymphocytes in vitro. Peripheral blood lymphocytes from heparinised venous blood of healthy subjects (n=17) were examined. Five and 10 mg ml-1 phytohemagglutinin (PHA) and 5 mg ml-1 pokeweed mitogen (PWM) were added. Depending on the concentration, calyx infusion of H. sabdariffa decreased mitogen-induced blastogenesis in normal subjects. It did not affect, however, the level of spontaneous proliferation of human lymphocytes in vitro. The infusion of hibisci flos may alleviate over-stimulated condition of the immune system in hypersensitivity ...

Fatal infection in mice after injection of immunosuppressive serum fractions from surgical patients.

Following surgical or accidental trauma many patients show suppression of cellular immunity. In this investigation sera from severely burned patients and patients undergoing aortic aneurysm repair were studied. Sera shown to suppress phytohaemagglutinin-induced blastogenesis of normal human lymphocytes were fractionated using ion exchange and G25 Sephadex chromatography. Suppressive activity was largely confined to a low molecular weight (LMW) fraction and was dose dependent. LMW fractions of normal sera had no significant suppressive activity. The purpose of this study was to test the causal relationship between immunosuppressive serum and decreased resistance to bacterial infection. Listeria monocytogenes infected mice were used as an in vivo model to test suppression of cellular immunity. Injection of LMW fractions of suppressive sera significantly increased mortality in these mice, but had no effect on non-infected mice. There was good correlation between the in vitro and in vivo effects of the suppressive fractions. These results suggest that a circulating factor in the serum of injured patients suppresses cellular immunity and may be responsible for impaired resistance to infection.

IgE-Dependent Activation of T cells by Allergen in Atopic Dermatitis: Pathophysiologic Relevance

The importance of interactions between allergen and IgE in allergen-mediated activation of T lymphocytes from patients with atopic dermatitis (AD) is unclear. A role for this interaction is implied by past evidence for IgE-facilitated presentation of allergen to T cells, but this phenomenon has only been demonstrated in specific in vitro systems biased to maximize the effect. It is therefore not known whether the process is relevant in patients. We now show that the responses to allergen of unmodified peripheral blood mononuclear cells (PBMC) from individual AD patients are significantly greater in the presence of fresh, unheated, IgE-containing autologous serum than the same serum heated under IgE-denaturing conditions or specifically depleted of IgE by immunoprecipitation. In six independent experiments, 59%-67% of the maximal in vitro PBMC response to allergen was found to be dependent upon the presence of IgE in autologous serum used at 5% final concentration. These data provide the first evidence that sufficient amounts of allergen-specific IgE and allergen-reactive T cells occur concomitantly in the blood of individual AD patients to allow IgE-enhanced T cell responses to allergen. We conclude that IgE-enhanced T cell responses are pathophysiologically relevant and a therapeutic target in AD.

Pharmacological effects of urinary products obtained after treatment with saiboku-to, a herbal medicine for bronchial asthma, on type IV allergic reaction.

To define the anti-allergic components in Saiboku-To, a herbal medicine for bronchial asthma, we examined the effects of 11 compounds found in post-administrative urine of Saiboku-To on concanavalin A-induced human lymphocyte blastogenesis in vitro and picryl chloride (PC)-induced mouse ear swelling in vivo. The urinary products of Saiboku-To were flavonoids and lignans derived from the constitutional herbs and their hydrogenated metabolites. Medicarpin derived from Glycyrrhiza glabra, magnolol and 8,9-dihydroxydihydromagnolol from Magnolia officinalis, baicalein, wogonin and oroxylin A from Suctellaria baicalensis inhibited lymphocyte blastogenesis in dose-dependent fashion with IC50 values ranging from 3.0 to 7.7 micrograms/mL, which corresponded to 20-100 times that of prednisolone IC50 (0.08 microgram/mL). Davidigenin, dihydrowogonin and dihydrooroxylin A, which are hydrogenated metabolites of liquiritigenin, wogonin and oroxylin A, respectively, had no or little effects on lymphocyte blastogenesis. Oral administration of Saiboku-To, medicarpin, baicalein, magnolol and baicalin (100 mg/kg), inhibited PC-induced ear swelling significantly by 23.5, 40.1, 30.5, 23.6 and 20.9%, respectively, though the effects were weaker than that of 5 mg/kg of prednisolone (52.9%). The results suggested that flavonoids and lignans tested in the present study were implicated in anti-asthmatic effect of Saiboku-To through suppression of type IV allergic reaction.

Suppression by azelastine hydrochloride of NF-kappa B activation involved in generation of cytokines and nitric oxide.

The influence of the anti-allergy agent azelastine hydrochloride (Azeptin) on NF-kappa B activation associated with the generation of cytokines and nitric oxide (NO) was investigated in various kinds of human and mouse cells. Azeptin dose-dependently suppressed both DNA and protein synthesis in human gingival fibroblasts (HF) and also suppressed blastogenesis of human peripheral blood lymphocytes (PBL). Generation of tumor necrosis factor-alpha, interleukin 1-beta, granulocyte-macrophage colony-stimulating factor and interleukin-6 from 10(-5) M Azeptin-treated PBL and human monocytes (HM) was decreased to approximately 1/3 to 2/3 of the control levels. In parallel with the decreased cytokine generation, each cytokine mRNA was less expressed in the presence of 10(-5) M Azeptin. In addition, both inducible nitric oxide synthase-mRNA level and NO generation in mouse peritoneal macrophages were suppressed by 10(-5) M Azeptin. Being compatible with those results, Azeptin (10(-5) M) suppressed activation of NF-kappa B in PBL, HM and HF. These results appear to indicate that suppression of cytokine and NO generation by Azeptin results at least partially from the inhibition of NF-kappa B activation.

Suppression by Azelastine Hydrochloride of NF-κB Activation Involved in Generation of Cytokines and Nitric Oxide

The influence of the anti-allergy agent azelastine hydrochloride (Azeptin®) on NF-κB activation associated with the generation of cytokines and nitric oxide (NO) was investigated in various kinds of human and mouse cells. Azeptin dose-dependently suppressed both DNA and protein synthesis in human gingival fibroblasts (HF) and also suppressed blastogenesis of human peripheral blood lymphocytes (PBL). Generation of tumor necrosis factor-alpha, interleukin 1-beta, granulocyte-macrophage colony-stimulating factor and interleukin-6 from 10−5 M Azeptin-treated PBL and human monocytes (HM) was decreased to approximately 1/3 to 2/3 of the control levels. In parallel with the decreased cytokine generation, each cytokine mRNA was less expressed in the presence of 10−5 M Azeptin. In addition, both inducible nitric oxide synthase-mRNA level and NO generation in mouse peritoneal macrophages were suppressed by 10−5 M Azeptin. Being compatible with those results, Azeptin (10−5 M) suppressed activation of NF-κB in PBL, HM and HF. These results appear to indicate that suppression of cytokine and NO generation by Azeptin results at least partially from the inhibition of NF-κB activation.

Function and expression of a human idiotypic network in Schistosomiasis japonica.

The cross-reactive idiotype (Hu-SJ-CRIM) is defined by polyclonal human anti-idiotypic antibodies derived from chronically S. japonicum infected patients. The present study shows that serum levels of Hu-SJ-CRIM expressed by antibodies to S. japonicum soluble egg antigen (SEA) are associated with acute infection and hepatosplenic disease. Xenogeneic anti-idiotypic antisera (anti-Hu-SJ-CRIM) suppressed human lymphocyte blastogenesis to SEA in vitro by 47-82% (P < 0.05). These anti-idiotypic antibodies also suppressed in vitro granuloma formation induced by SEA coated heads in a dose dependent manner. This immunosuppression was antigen specific in that mitogen (PHA) or non-related antigen (PPD) induced blastogenic responses were not suppressed. Surprisingly, anti-idiotypic antibodies (anti-SJ-CRIM), which describe the mouse correlate CRIM were not suppressive in the human blastogenesis or in vitro granuloma formation assays. These data indicate a dichotomy in the function and specificity of the idiotype/anti-idiotype human and murine immune networks in S. japonicum infection. Thus, only the patient derived molecules and serology form the basis for an immunoregulatory network in Schistosomiasis japonica.

Natural flavonoids and lignans are potent cytostatic agents against human leukemic HL-60 cells

Anti leukemic-cell efficacy of 28 naturally occurring and synthetic flavonoids and 11 naturally occurring lignans on human promyelocytic leukemic cell line HL-60 were examined using MTT assay methods. Differences between anti cell-proliferative activity and cytotoxicity of these compounds were compared with thos of 4 clinical anti-cancer agents. Eight of the 28 flavonoids and 4 of the 11 lignans showed considerable suppressive effects on HL-60 cell growth with IC 50s ranging from 10–940 ng/ml. Among these compounds, genistein, honokiol, machilin A, matairesinol, and arctigenin had the strongest effects with IC 50s less than 100 ng/ml, which were almost equivalent to the effects of current anti-cancer agents. The flavonoid genistein and the lignans, however, showed little or no cytotoxicity against HL-60 cells as assessed by dye exclusion tests (LC 50s>2,900ng/ml), whereas the regular anti-cancer agents had potent cytotoxicity. All of the flavonoids and lignans, except for machilin A and arctigenin, were less effective against growth of human T lymphocytic leukemia cell line MOLT- 4. In addition, the flavonoid and the lignans showed little or no inhibiting activity on mitogen-induced blastogenesis of human peripheral-blood lymphocytes. The lignans and genistein were strongly suppressive against incorporations of [ 3H]thymidine, [ 3H]uridine, and [ 3H]leucine into HL-60 cells. These results showed that some of the naturally occurring flavonoids and lignans inhibited HL-60 cell growth with a non-toxic mechanism, possibly via cessation of DNA, RNA, and/or protein synthesis of the leukemic cells.

Reduced human lymphocyte blastogenesis and enhancement of adenosine triphosphate (ATP) by L-carnitine.

Carnitine is associated with lipid synthesis and its deficiency may lead to cardiomegaly with parenchymal lipid in the heart, kidney and liver. In our study we found that pretreatment of peripheral blood mononuclear cells (PBMC) with serial dilutions of L-Carnitine (100 micrograms/ml-1 pg/ml) inhibits, in a dose-dependent manner, lymphocyte DNA synthesis stimulated with PHA (20 micrograms/ml). L-Carnitine did not have any effect on resting PBMC. The maximum inhibition was found at 10 micrograms/ml of L-Carnitine. Moreover, in a time-course study and using an enzymatic analysis (ATP monitoring reagent), L-Carnitine enhanced ATP production on PBMC treated and untreated with PHA, reaching a maximum effect at 30 min incubation. In another set of experiments PBMC were treated with L-Carnitine alone and in combination with PHA, and the percent of receptors CD3, CD4, and CD8 were calculated with flow cytometry. After the cell incubation with L-Carnitine, the percent of all receptors studied did not change compared to L-Carnitine-untreated cells (controls). These data suggest that L-Carnitine inhibits, in a dose-dependent manner, lymphocyte blastogenesis induced by PHA, probably through the enhancement of ATP synthesis, which is considered an inhibitor of phospholipase C activity and a suppressor in lymphocyte cultures.

Suppression of lymphocyte adenosine 3':5'-cyclic monophosphate (cAMP) by delta-9-tetrahydrocannabinol.

Delta-9-tetrahydrocannabinol (THC) is the major psychoactive component of marijuana. Suppression of mitogen-stimulated blastogenesis of human lymphocytes in vitro by THC was previously demonstrated. This effect was shown to be concentration dependent with the non-toxic concentrations 5, 7.5, and 10 micrograms THC/ml showing the greatest suppression. However, the mechanism(s) by which THC induces suppression are still unclear. The current study examines the effect of THC on the adenosine 3':5'-cyclic monophosphate (cAMP) pathway second messenger system, which is involved in activation of human peripheral blood lymphocytes. Lymphocyte cAMP levels were stimulated using three hormone receptor stimulators, isoproterenol, histamine, or 5'-N-ethylcarboxamide adenosine (NECA), each of which utilizes a different receptor to enhance cAMP production. THC suppressed cAMP levels independently of the hormone and receptor utilized. Levels of cAMP in non-mitogen-stimulated peripheral blood mononuclear cells and plastic non-adherent lymphocytes, as well as cells stimulated with phytohemmagglutinin, were suppressed by THC. Suppression of cAMP production by THC was further examined to determine whether inhibition involved a GTP-binding protein (Gi), which is known to down-regulate cAMP production. Cells were pre-treated with pertussis toxin to inhibit Gi activity; this blocked the THC-induced suppression of cAMP production. These results suggest that THC can exert its effects on second messenger systems at the lymphocyte membrane level, and that a pertussis toxin-sensitive Gi protein may be involved. Thus, second messenger regulated pathways may be involved in THC-induced immune suppression. However, the relationship between alteration of cAMP production and suppression of lymphocyte function due to the presence of THC in the medium remains to be established.

Human EBV-transformed lymphocytes of patients with Schistosoma japonicum infection secrete idiotypically related immunoregulatory antibodies

Lymphocytes derived from the peripheral blood of individuals infected with Schistosoma japonica were transformed in vitro with Ebstein-Barr virus (EBV). Serological characterization of antibody molecules revealed both antigen reactive (idiotypic) and anti-idiotypic transformants. One idiotypic EBV transformant, LO2C2, describes a major cross-reactive idiotype associated with anti-antigen binding molecules. Other antibody populations expressing idiotypic cross-reactivity were derived from separate individuals showing shared idiotypy in S. japonicum field study populations in the Republic of Philippines. Both idiotypic and anti-idiotypic molecules suppressed parasite antigen-driven blastogenesis of heterologous human peripheral blood lymphocytes. The data show a serologically related immunoregulatory immune network in patients in the Republic of the Philippines which is serologically distinct from idiotypy expressed in other selected S. japonicum endemic areas in the Far East.

Promotion of lymphocyte blastogenesis by hemodialysate of chronic renal failure.

Effects of hemodialysate of patients with chronic renal failure (CRF) on blastogenesis of human peripheral blood lymphocytes in the presence of concanavalin A or phytohemagglutinin was investigated. Hemodialysates from 11 CRF patients significantly promoted lymphocyte blastogenesis when compared with control dialysis fluids (p less than 0.01). The strongest activity (39% promotion of the lymphocyte blastogenesis) was observed with a hemodialysate obtained at 0.5 h after beginning dialysis. The activity decreased thereafter. On the contrary, the blastogenesis-promoting activity in plasma decreased significantly after hemodialysis (p less than 0.01, n = 11). To further confirm the presence of low-molecular-weight factor(s) in CRF, ultrafiltration of plasma obtained from CRF and healthy subjects was conducted using a membrane filter with a molecular cutoff of 3,000 D. The filtrate of CRF plasma significantly promoted lymphocyte blastogenesis when compared to that of healthy subjects (p less than 0.01). Heat treatment (100 degrees C, 40 min) did not abolish the activity of the hemodialysate. None of the drugs taken by the patients nor creatinine accumulated in CRF promoted the lymphocyte blastogenesis. Chromatographic analysis of a hemodialysate demonstrated several peaks which were absent in the control dialysis fluid. These results showed the presence of a novel lymphocyte-stimulating factor(s) in CRF, which is heat stable and has a low molecular weight (less than 3,000 D).

Interleukin-2 (IL-2) production and interleukin-2 receptor expression in patients with aplastic anemia

IL-2 production and IL-2 receptor (Tac antigen) of the peripheral blood mononuclear cells in 30 patients with aplastic anemic (AA) were studied. We found that mononuclear cells from patients produce spontaneously IL-2 in the absence of exogenous lectin stimulation, the proportion of Tac+ cells in mononuclear cells increased. The release of IL-2 and/or Tac antigen expression were elevated in almost every patient with AA. The plasma from patients stimulate mitogen-induced blastogenesis and Tac antigen expression of normal human lymphocytes. Immunological 1 abnormalities of patients with AA possibly might represents secondary response to bone marrow depression.

The Influence of FK‐506 and Low‐Concentration Ciclosporin on Human Lymphocyte Activation Antigen Expression and Blastogenesis: a Flow Cytometric Analysis

The influence of FK-506 and ciclosporin (CsA), either alone or in combination, on PHA- and alloantigen-induced human blood lymphocyte transformation was investigated. The concentrations of FK-506 and CsA required to cause 50% inhibition of mitogen-induced thymidine incorporation were 1.0 and 200 ng/ml respectively. Evidence of synergistic inhibition of DNA synthesis was obtained when doses of each drug below these concentrations were combined (FK-506, less than or equal to 0.5 ng/ml; CsA less than or equal to 50 ng/ml). In contrast, FK-506 and CsA failed to inhibit PHA-induced increases in cell size and granularity determined by flow cytometric analysis at 1 and 3 days of culture. Moreover, no significant changes in the expression of the interleukin 2 receptor (IL-2R; CD25), transferrin receptor (TR; CD71) or HLA-DR were observed in terms of percentage positive lymphocytes or mean cell surface membrane fluorescence intensity. In primary mixed lymphocyte cultures, 50% inhibition of thymidine incorporation was obtained with FK-506 and CsA concentrations of approximately 0.25 and 25 ng/ml respectively. Evidence of synergy was obtained when lower doses of each drug were combined (FK-506, less than or equal to 0.1 ng/ml; CsA less than or equal to 2 ng/ml). On their own, these concentrations of CsA were ineffective. In contrast to corresponding PHA-stimulated cells, FK-506-treated alloactivated lymphocytes exhibited reductions in IL-2R, TR and HLA-DR antigen expression, which in the cases of IL-2R and TR were further reduced by combination of FK-506 with low-concentration CsA. These data provide further evidence of the potential of combined low-dosage FK-506 and CsA for the control of human T-cell activation and proliferation in response to allostimulation.

A cytokine, human lymphocyte blastogenesis inhibitory factor (LBIF): Structure and functions: [formula omitted] Institute of Immunological Science, Hokkaido Univ. Sapporo 060 Japan

A cytokine, human lymphocyte blastogenesis inhibitory factor (LBIF): Structure and functions: [formula omitted] Institute of Immunological Science, Hokkaido Univ. Sapporo 060 Japan

Synthetic peptides homologous to HIV transmembrane glycoprotein suppress normal human lymphocyte blastogenic response

Human immunodeficiency virus (HIV) envelope glycoprotein is synthesized as a polyprotein precursor of 160 kDa (gp 160) and is subsequently cleaved into an amino terminus subunit, gp 120, and a carboxyl terminus transmembrane subunit, gp 41. Two synthetic peptides corresponding to amino acid sequences 735–752 and 846–860, respectively, as deduced from the nucleotide sequence of HTLV-III B gp 160 were synthesized and used to assess their effects on normal human lymphocyte blastogenesis. Peptides 735–752 and 846–860 conjugated to protein carriers, but not free peptides, exerted a pronounced suppression of the normal human lymphocyte proliferative response to concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM), and alloantigens. A synthetic peptide homologous to a 17 amino acid sequence of the gene product of HIV trans-acting transcriptional (TAT III) gene had no suppressive effects. Peptides 735–752 and 846–860 also inhibited the IL-2-dependent proliferation of the murine CTLL-2 cell line and the PHA-induced proliferation of normal mouse spleen cells. HIV peptide-induced suppression of human blastogenesis required a 2- to 3-day incubation of responder cells with peptides, suggesting that binding of peptides to the cell membrane was not sufficient for suppression. These results suggest that, in addition to the selective cytopathic effects of HIV, the etiological agent of the acquired immunodeficiency syndrome (AIDS), on the T-helper/inducer lymphocyte subset, viral peptide-mediated immunosuppression may also play an important role in the pathogenesis of the disease. Moreover, these data clearly indicate the need to address the potential immunosuppressive property of HIV antigens in the effort to select and develop effective prophylactic means against AIDS.

Studies on the mechanism of T cell inhibition by the Pseudomonas aeruginosa phenazine pigment pyocyanine.

Pseudomonas aeruginosa and its products have been shown to inhibit mitogen-induced human lymphocyte blastogenesis as measured by [3H]TdR uptake. The phenazine pigment pyocyanine has been identified as one of the inhibitors present in cellfree culture supernatants. To determine the mechanism of the inhibitory action of pyocyanine, we studied its effect on the early stages of T cell activation. Pyocyanine inhibited lymphocyte stimulation induced by specific antigens, the lectin concanavalin A and the calcium ionophore, ionomycin, suggesting that its inhibitory effect is not dependent on interference with the T cell antigen receptor complex itself. Using quin-2, we showed that pyocyanine did not interfere with the mitogen-induced increase in cytosolic-free Ca2+. We also showed that pyocyanine did not interfere with the function of calmodulin stimulated Ca2+-Mg2+ ATPase activity, indicating that the mechanism of action of pyocyanine differs from that of the structurally related phenothiazine compounds. Analysis of IL 2 production and IL 2 receptor expression clearly showed that pyocyanine inhibits the production of this essential lymphokine as well as the expression of IL 2 receptors on the T cell membrane. This inhibition is dose dependent and not due to cellular toxicity. There was parallel inhibition of growth in cell volume as well as [3H]TdR uptake. Thus, our results demonstrate that pyocyanine inhibits T cell proliferation by decreasing the production of the critical lymphokine IL 2 and by decreasing the expression of the IL 2 receptor. Local suppression of lymphocyte stimulation by phenazine pigments such as pyocyanine may interfere with cellular immune responses that may be necessary for eradication of chronic infection with P. aeruginosa.

Inhibitory effects of tunicamycin on the mixed lymphocyte reaction and mitogen-induced lymphocyte blastogenesis

The role of asparagine(Asn)-linked saccharides on the surface of lymphocytes in cellular interactions was examined by performing studies on the effects of tunicamycin (TM), which inhibits the glycosylation of proteins N-glycosylated at asparagine residues, on the mixed lymphocyte reaction (MLR) and mitogen-induced lymphocyte blastogenesis of human lymphocytes by measuring 3H-thymidine (TdR) incorporation. Responses were expressed as percentages of that of the control (MLR without TM). The lymphocyte blastogenesis in the one- and two-way MLR were, respectively, 43.1% and 48.0% of the control at 0.1 μg/ml of TM, and 5.5% and 7.2% at 1 μg/ml of TM. The inhibitory effect of TM on the one-way MLR was shown using TM-pretreated stimulator cells, TM-pretreated responder cells or both. TM blocked lymphocyte blastogenesis in the secondary as well as in the primary MLR. The inhibitory effect of TM on the two-way MLR was observed when TM was added on day 0 to day 2, but not on day 4 of incubation. TM blocked mitogen-induced lymphocyte blastogenesis by phytohemagglutinin, concanavalin A or by pokeweed mitogen. As TM had no cytotoxic effect on cultured cells, these inhibitory effects of TM were thought to be due to the loss of Asn-linked saccharides from glycoprotein of the surface of lymphocytes. These findings indicated that Asn-linked saccharides of glycoprotein on the surface of lymphocytes were important in cellular interactions that are necessary for the cellular immune response.

Increase in incorporation into lymphocytesin vitro of esterified anti-inflammatory corticosteroids

Corticosteroids esterified at C-17 and C-21 such as hydrocortisone 17-butyrate 21-propionate (HBP) and prednisolone 17-valerate 21-acetate (PVA) inhibited PHA-induced blastogenesis of human blood lymphocytes more intensively than hydrocortisone (HC) and prednisolone (PSL), the parent corticosteroids. When lymphocytes were incubated with HBP or HC, the total amount of HBP incorporated into lymphocytes was much larger than that of HC. The amount of HBP bound specifically to the receptors was also increased, which may be due to the increased concentration of corticosteroid in lymphocytes. These results suggest that the increased incorporation of the esterified corticosteroids into cells would be most important to produce an intensive biological activity.

Influence of ofloxacin, norfloxacin, nalidixic acid, pyromidic acid and pipemidic acid on human gamma-interferon production and blastogenesis.

Several new quinolone derivatives were investigated for their influence on human lymphocyte blastogenesis and gamma-interferon production following concanavalin A stimulation. All the antimicrobials induced inhibition of lymphocyte DNA synthesis. The gamma-interferon measurements showed that nalidixic acid and norfloxacin have a negative influence on lymphokine production and release.