Effects of phospholipid platelet activation factor on proliferation of B-lymphocytes of the peripheral blood of healthy donors and patients with bronchial asthma

The influence of the phospholipid platelet activation factor (PAF) was studied on pokeweed mitogen (PWM)-stimulated proliferation of peripheral blood lymphocytes in patients with bronchial asthma and normal subjects. It was found that PAF exerts dose-dependent activation with following suppression mainly on B-cells after its action on the whole population of lymphocytes activated by PWM. Besides, the influence of PAF on lymphocyte proliferation seemed to be mediated by monocytes since removal of monocytes from the whole population of mononuclear cells abolished the lymphocyte activation induced by PWM. Indomethacin inhibits lymphocyte proliferation activation induced by PAF. The results indicate that PAF has an effect on the IgE and IgG synthesis by blood B-lymphocytes in patients with bronchial asthma.     

Glutathione modulates activation-dependent proliferation of human peripheral blood lymphocyte populations without regulating their activated function

L-Buthionine-(S,R)-sulfoximine (BSO) specifically depletes GSH synthesis by inactivating gamma-glutamylcysteine synthetase, whereas 2-ME augments intracellular GSH concentration. These reagents were used to examine GSH regulation of the proliferation and function of human PBL in response to IL-2 or OKT-3 mAb directed at the CD3 T cell Ag. 2-ME enhanced both IL-2-induced proliferation of PBL and CD3- large granular lymphocytes (LGL) and OKT-3 mAb-induced proliferation of CD3+ T cells. BSO partially suppressed activation-induced proliferation in CD3- LGL and CD3+ T cells and totally inhibited the positive co-proliferative regulation by 2-ME in these cells. By contrast, neither BSO nor 2-ME appeared to affect the activation-dependent differentiation of cytotoxic lymphocytes. The absence of effect of 2-ME or BSO on activation-induced PBL NK activity and T cell cytotoxic potential was supported by their negligible effect on the induction of two different markers of activated cytotoxic lymphocytes, namely pore-forming protein gene expression and benzoyloxycarbonyl-1-L-lysine thiobenzylester-esterase activity. BSO inhibition of CD3- LGL proliferation accounted for the inhibitory effects of BSO on both IFN-gamma production in IL-2-stimulated PBL cultures and IL-2-induced PBL lymphokine activated killer activity. The modulatory effects of 2-ME and BSO on lymphocyte proliferation regardless of phenotype (LGL vs T cell) or stimulation (IL-2, via CD3, lectin, etc.) and the functional differentiation of cytotoxic lymphocytes independent of proliferation suggests that these cells share a common site of GSH regulation close to or at the level of DNA synthesis.     

Measles virus-induced suppression of lymphocyte proliferation

The mechanism by which measles virus induces immunosuppression was investigated using an in vitro system employing phytohemagglutinin (PHA)-induced human peripheral mononuclear cell (PBMC) proliferation. At a multiplicity of infection of 1.0 or greater measles virus significantly inhibited (45%) the proliferation of PBMC. This inhibition was not due to an alteration in the kinetics of proliferation. PHA-stimulated PBMC were then infected with measles virus for 72 hr and irradiated (3200 rad) to prevent further proliferation. These infected, irradiated PBMC when added to fresh autologous PBMC caused significant inhibition of lymphoproliferation over a wide range of infected:fresh cell ratios (maximum inhibition seen at a 1:1 ratio, 85% inhibition). Virus recovered from the irradiated, infected cells was 100-fold lower than the virus titer needed to cause inhibition by direct addition of measles virus. However, antibody to measles virus reversed the inhibition. Virus-free supernatant fluids from the infected irradiated cells caused immunosuppression of the PHA response. This immunosuppressive material induced by the measles virus was maximally produced after 72 hr and did not appear to require viral replication. This factor was not prostaglandin E or interferon-alpha or -gamma. The production of such suppressive factors during viral infection may explain some of the profound immunosuppression seen in situations in which little or no infectious virus can be detected.     

Role of interleukin 2 on enhancement of concanavalin A-induced human peripheral blood lymphocyte proliferation by murine B cell mitogens

Murine B cell mitogens such as bacterial lipopolysaccharide (LPS), butanol-extracted water soluble adjuvant (Bu-WSA), dextran sulfate (DS), synthetic muramyl dipeptide (MDP), and its analog MDP-Lys (L18) do not show any mitogenic ability in vitro on human peripheral blood lymphocytes or mixed cell populations of purified T and B cells obtained from the lymphocytes in an ordinary culture system. However, these mitogens are capable of enhancing the mitogenic effect of concanavalin A (Con A) in the cultures. In the presence of one of these mitogens, the activity of interleukin 2 (IL 2), but not interleukin 1, in the supernatants obtained from cultures containing Con A-stimulated T cell and B cell populations was higher than that of control cultures. The role of the newly produced IL 2 in the synergistic effect of the mitogens in human lymphocyte cell cultures was discussed.     

A Rho exchange factor mediates fMet-Leu-Phe-induced NF-kappaB activation in human peripheral blood monocytes

We reported previously that fMLP stimulates NF-kappaB activation, and this function of fMLP requires small GTPase RhoA in human peripheral blood monocytes (Huang, S., Chen, L.-Y., Zuraw, B. L., Ye, R. D., and Pan, Z. K. (2001) J. Biol. Chem. 276, 40977-40981). Here we present evidence that RhoA associates specifically with the guanine nucleotide exchange factor Lbc in human peripheral blood monocytes stimulated with fMLP and that Lbc specifically catalyzes the guanine nucleotide exchange activity of RhoA in human peripheral blood monocytes. Cotransfection of the monocytic THP1 cells with lbc with a kappaB promoter reporter plasmid results in a marked increase in NF-kappaB-mediated reporter gene expression. Finally, Lbc-enhanced NF-kappaB activation is inhibited by a RhoA inhibitor, C3 transferase from Clostridium botulinum. A dominant-negative form of RhoA (T19N) also inhibited Lbc-enhanced reporter gene expression in a kappaB-dependent manner. These results indicate that guanine nucleotide exchange factor Lbc is a novel signal transducer for RhoA-mediated NF-kappaB activation in human peripheral blood monocytes stimulated with bacterial products.     

Human peripheral blood T lymphocyte proliferation after activation of the T cell receptor: effects of unsaturated fatty acids

Oils enriched in certain polyunsaturated fatty acids suppress joint pain and swelling in rheumatoid arthritis patients with active synovitis. Because T lymphocyte activation is important for propagation of joint tissue injury in patients with rheumatoid arthritis, we examined the effects of fatty acids added in vitro on proliferation of human T lymphocytes stimulated with monoclonal antibodies to CD3 and CD4. Unsaturated fatty acids reduced T cell proliferation in a dose dependent manner (dihomogammalinolenic acid > gammalinolenic acid > eicosapentaenoic acid > arachidonic acid). Removal of fatty acids from cultures before cell stimulation did not change the effects, but addition of fatty acids after cell stimulation failed to reduce T cell responses. The saturated palmitic acid did not influence T cell growth. These studies indicate that small changes in cellular fatty acids can have profound effects on early events in T cell signaling and on T cell function.     

Heterogeneous accessory cell requirement for human peripheral blood T lymphocyte activation by PHA into IL-2-responsive colony-forming cells

Mitogen-driven T cell proliferation in liquid culture requires accessory cells that cooperate in interleukin 2 production. We have investigated the accessory cell requirement for human lymphocyte colony formation under PHA stimulation. Semisolid medium limits cell-to-cell contact emphasizing the role of cooperating cells both in growth factor production and in triggering events. Culturing at high T cell density demonstrates that accessory cells can be substituted for colony formation by exogenous IL-2. Culturing at low T cell density in the presence of IL-2 also demonstrates that accessory cells are required for activation of a subset of progenitors into IL-2 responsive colony-forming cells. Consequently, T colony progenitors, contained in the E-rosetting cell fraction of peripheral blood, are heterogeneous in their triggering signals: a minor subset is directly inducible by PHA, and a major subset is inducible by PHA in the presence of accessory cells. We found that monocytes and some leukemic B cells support effective accessory function in both colony growth factor production and colony progenitor sensitization.     

Platelet-activating factor (PAF) receptor antagonists inhibit mitogen-induced human peripheral blood T-cell proliferation

The addition of L-652,731 and L-653,150, two synthetic PAF-specific receptor antagonists, to 72 hour cultures of phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear leukocytes (PBML) caused a dose-dependent inhibition of (3H)-thymidine incorporation into T-cells (IC50: 25 microM and 3.2 microM, respectively). This inhibition was not reversed by exogenous interleukin (IL)-1 and IL-2. PAF receptor antagonists did not affect the expression of IL-2 receptors (TAC-antigen) on T-cells. Exogenous PAF which by itself had no significant effect on PHA-stimulated PBML proliferation, only partially reversed the inhibition of proliferation caused by PAF receptor antagonists. These results may suggest the involvement of endogenously produced PAF in the regulation of immune reactions.     

Lymphoblastoid cell-induced suppression of human peripheral blood leukocyte mitogenic responses

Two lymphoblastoid tumor cell lines, the Burkitt lymphoma derived BJAB cell line which is free of Epstein-Barr virus (EBV) and B95-8 cells, which are marmoset lymphocytes transformed by EBV isolated from an infectious mononucleosis patient, were studied in regards to their effects on the blastogenic responsiveness of normal human peripheral blood leukocytes stimulated in vitro with mitogens. Mitomycin C treated tumor cell suspensions, when cocultured with normal human blood leukocytes, markedly depressed the expected blastogenic responses in vitro to concanavalin A, pokeweed mitogen, and phytohemagglutin. In addition, cell-free sonicates from the cell lines also depressed blastogenic responsiveness of the leukocytes in vitro. Heating the sonicates for 10 min at 100 degrees C markedly diminished the suppressive properties of the sonicates, as did ultraviolet light irradiation. The suppressive activity of the B95-8 sonicates was pelleted by high speed centrifugation as compared to the activity of sonicates derived from the BJAB cells. Further studies are warranted to determine the nature and mechanism of suppression of blastogenic responsiveness of normal human leukocytes by soluble components derived from such lymphoblastoid cell lines.     

Tumor necrosis factor alpha up-regulates non-lymphoid Fas-ligand following superantigen-induced peripheral lymphocyte activation

Members of the tumor necrosis factor (TNF) and TNF receptor families play important roles in inducing apoptosis and mediating the inflammatory response. Activated T lymphocytes can trigger the expression of Fas-ligand on non-lymphoid tissue, such as intestinal epithelial cells (IEC), and this, in turn, can induce apoptosis in the T cells. Here, we examine the role of TNFalpha in this feedback regulation. Injection of TNFalpha into mice caused a rapid up-regulation of Fas-ligand mRNA in IEC. TNFalpha-induced activation of the Fas-ligand promoter in IEC requires NF-kappaB as this was blocked by an I-kappaBalphaM super-repressor and by mutation of an NF-kappaB site in the Fas-ligand promoter. Activation of T cells by antigen induced Fas-ligand expression in IEC in vivo in wild type, but not in TNFalpha-/- or TNFR1-/- mice. These results define a novel pathway wherein TNFalpha, produced by activated T cells in the intestine, induce Fas-ligand expression in IEC. This is the first observation that one member of the TNF superfamily mediates the regulation of another family member and represents a potential feedback mechanism controlling lymphocyte infiltration and inflammation in the small intestine.     

Separation and purification of lymphocyte chemotactic factor (LCF) and interleukin 2 produced by human peripheral blood mononuclear cells

Two T-cell chemotactic factors, lymphocyte chemotactic factor (LCF) and interleukin 2 (IL-2), were separated and characterized from culture supernatants of concanavalin A-stimulated human peripheral blood mononuclear cells. LCF was purified approximately 7800-fold to homogeneity from culture supernatant using gel filtration and high-performance liquid chromatography (HPLC). LCF was found to be distinct from both IL-2 and interleukin-1. Sephadex G-100 gel filtration of crude supernatants from concanavalin A-stimulated mononuclear cells showed two molecular weight regions of T lymphocyte chemotactic activity. A 10,000- to 25,000-Da region contained both IL-2 and LCF and a 45,000- to 75,000-Da region contained only a high molecular weight form of LCF. Both high and low molecular weight species of LCF eluted with 40-44% acetonitrile from a reversed-phase C18 HPLC column. IL-2 present only in the low molecular weight region eluted from the C18 column with 65-75% acetonitrile. The migration of T lymphocytes to IL-2 was totally inhibited by anti-interleukin 2 receptor antibody while the response of T cells to LCF was unaffected. LCF eluting off the C18 column was purified to homogeneity by two subsequent cycles of gel filtration HPLC. The resultant protein showed a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular weight of 10,500. The data presented here demonstrate that IL-2 and LCF are distinct lymphocyte chemotactic factors and although they are not readily separable from crude supernatants by molecular sieve chromatography, they can easily be distinguished by reversed-phase HPLC.     

Regulation of human lymphocyte proliferation by a heterodimeric cytokine, IL-12 (cytotoxic lymphocyte maturation factor).

IL-12 is a heterodimeric cytokine that was identified on the basis of its ability to synergize with IL-2 in the induction of cytotoxic effector cells and was originally called cytotoxic lymphocyte maturation factor (CLMF). IL-12 was also found to stimulate the proliferation of PHA-activated lymphoblasts which were greater than 90% CD3+ T cells. In this report we further characterize the effects of IL-12 on lymphocyte proliferation. Studies with purified subpopulations of PHA-activated lymphoblasts and with cloned lines of human T cells indicated that IL-12 caused the proliferation of activated T cells of both the CD4+ and CD8+ subsets. This effect of IL-12 was independent of IL-2 because it was not blocked by antibodies to either IL-2 or IL-2R. The maximum proliferation induced by IL-12 was 31 to 72% of the maximum caused by IL-2; however, IL-12 was active at a lower effective concentration (EC50 = 8.5 +/- 1.3 pM) than IL-2 (EC50 = 52 +/- 8 pM). Combination of suboptimal amounts of IL-12 and IL-2 resulted in additive proliferation, up to the maximum induced by IL-2 alone. IL-12 also caused the proliferation of lymphocytes activated by culture with IL-2 for 6 to 12 days. CD56+ NK cells were among the IL-12-responsive cells in the IL-2-activated lymphocyte population. Unlike IL-2 or IL-7, IL-12 caused little or no proliferation of resting peripheral blood mononuclear cells (PBMC). In this regard, IL-12 was similar to IL-4. However, IL-12 could enhance the proliferation of resting PBMC caused by suboptimal amounts of IL-2, whereas IL-4 inhibited IL-2-induced PBMC proliferation. Thus, IL-12 is a growth factor for activated human T cells and NK cells; however, its spectrum of lymphocyte growth-promoting properties is distinct from that of IL-2, IL-4, or IL-7.     

Endorphins stimulate normal human peripheral blood lymphocyte natural killer activity

Opioid peptides are present in peripheral blood, and may bind to human lymphocytes. In order to determine their influence on human lymphocytes we studied the effect of endogenous opioid peptides on human lymphocyte natural killer function. Beta-endorphin and several analogues (i.e., gamma-endorphin) are shown to enhance human peripheral blood natural killer function. The enhancement of natural killing by these opioid peptides was dose-dependent and naloxone (an opiate antagonist) reversible. In studying various analogues of beta-endorphin, beta-lipotropin and gamma-endorphin were approximately 3-5 times more effective at enhancing peripheral blood NK function than Leu-enkephalin and -endorphin. In addition, we observed that naloxone reversed human fibroblast interferon mediated enhancement of human blood lymphocyte natural killer function. These observations suggest that circulating endogenous opioid peptides may have a physiologic role in regulating human blood lymphocyte natural killing.     

Inhibition of human cytotoxic T lymphocyte activity in vitro by autologous peripheral blood granulocytes

Peripheral blood granulocytes from normal healthy donors were found to reproducibly inhibit the cytolytic effector function of specifically sensitized cytotoxic T lymphocytes in vitro when co-incubated with these effector cells and target cells in 8 hr 51Cr release assays. Inhibition required intact granulocytes, was proportional to the number of granulocytes present, and was independent of granulocyte adherence, phagocytic function, and viability. Equivalent numbers of enriched normal or leukemic peripheral T lymphocytes did not cause inhibition of 51Cr release, and preincubation of granulocytes with effectors did not significantly alter viability or cytotoxic function. Because granulocytes can inhibit natural killer cell function in vitro, these data indicate that granulocytes can regulate diverse antigen-specific and spontaneous cytotoxic functions in vitro, suggesting that circulating granulocytes may have the potential for in vivo regulation of these cytotoxic effectors.     

Early increase in lymphocyte cyclic nucleotide phosphodiesterase activity upon mitogenic activation of human peripheral blood mononuclear cells.

Cyclic nucleotide phosphodiesterase activity of human peripheral blood mononuclear cells was significantly increased following a short (30 min) incubation with the mitogenic lectin Concanavalin A. Con A stimulated phosphodiesterase activity to the same extent whatever the subcellular compartment (homogenate, cytosol or pellet). Further separation of the Con A-activated mononuclear cells into lymphocyte-enriched and monocyte-enriched populations showed that the Con A-induced increase of phosphodiesterase activity exclusively affected the lymphocyte-enriched population. In lymphocytes, cyclic GMP phosphodiesterase activity was more importantly enhanced by Con A (+275%) than cyclic AMP phosphodiesterase activity (+75%). The increase of both activities occurred as early as from 10 min of Con A incubation and proved to be maximal with Con A doses of 2.5 and 5 micrograms per 10(6) cells, lower and higher doses being less effective. Inhibition experiments with reference inhibitors suggested that, among the high affinity phosphodiesterase isoforms, the cyclic GMP-inhibited enzyme might be more selectively enhanced by Con A than the cyclic AMP-specific, Rolipram-sensitive one. The non-mitogenic lectin Helix pomatia hemagglutinin, was not able to enhance cyclic nucleotide phosphodiesterase activity of human mononuclear cells whereas anti-CD3 monoclonal antibody, although being less effective than Con A, exhibited a significant stimulatory effect. Putting together these results suggest that the early increase in phosphodiesterase activity might be a normal step involved in the mitogenic activation of human lymphocyte.     

Regulation of human lymphocyte proliferation by fatty acids

In the present study, the effect of increasing concentrations of palmitic (PA, C16:0), stearic (SA, C18:0), oleic (OA, C18:1, n-9), linoleic (LA, C18:2n-6), docosahexaenoic (DHA, C22:6 n-3) and eicosapentaenoic (EPA, C20:5 n-3) acids on lymphocyte proliferation was investigated. The maximal non-toxic concentrations of these fatty acids for human lymphocytes in vitro were determined. It was also evaluated whether these fatty acids at non-toxic concentrations affect IL-2 induced lymphocyte proliferation and cell cycle progression. OA and LA at 25 microM increased lymphocyte proliferation and at higher concentrations (75 microM and 100 microM) inhibited it. Both fatty acids promoted cell death at 200 microM concentration. PA and SA decreased lymphocyte proliferation at 50 microM and promoted cell death at concentrations of 100 microM and above. EPA and DHA decreased lymphocyte proliferation at 25 and 50 microM being toxic at 50 and 100 microM, respectively. PA, SA, DHA and EPA decreased the stimulatory effect of IL-2 on lymphocyte proliferation, increasing the percentage of cells in G1 phase and decreasing the proportion of cells in S and G2/M phases. OA and LA caused an even greater pronounced effect. The treatment with all fatty acids increased neutral lipid accumulation in the cells but the effect was more pronounced with PA and DHA. In conclusion, PA, SA, DHA and EPA decreased lymphocyte proliferation, whereas OA and LA stimulated it at non-toxic concentrations.     

Inhibition of activation of human peripheral blood eosinophils by Y-24180, an antagonist to platelet-activating factor receptor.

We examined the effects of Y-24180, a potent and long-acting antagonist to platelet-activating factor (PAF) receptor, on the PAF- or leukotriene B4 (LTB4)-induced activation of eosinophils using human peripheral blood in vitro. As activation markers, CD11b expression level and soluble intercellular adhesion molecule-1 (sICAM-1)-binding activity were analyzed by flow cytometry. Y-24180 significantly inhibited PAF-induced increase in the ratio of strongly positive cells for CD11b expression and sICAM-1 binding at 0.01 microM or more. WEB 2086, another PAF receptor antagonist, also inhibited the increase significantly at 1 microM or more. LTB4-induced increases in the ratio of strongly positive cells for CD11b expression and sICAM-1 binding were inhibited by Y-24180 at 1 microM, but not WEB 2086 up to 10 microM. These results indicate that Y-24180 inhibits the PAF- or LTB4-induced activation of eosinophils in human peripheral blood more potently than WEB 2086.     

Inhibition of human lymphocyte proliferation and interleukin 2 production by platelet activating factor (PAF-acether): reversal by a specific antagonist, BN 52021

When added to a 72 h culture of human peripheral blood mononuclear leukocytes stimulated with phytohemagglutinin, PAF-acether caused a significant inhibition (40-65%) of proliferation at concentrations of 10(-8) to 10(-6) M. This inhibition was reversed by the specific PAF antagonist, BN 52021. It was also reversed by indomethacin, suggesting that PAF-acether mediated this suppression via cyclooxygenase metabolites of arachidonic acid. IL-2 production, measured at 24 h of lymphocyte proliferation, was similarly impaired (50-66%) by 10(-8)-10(-6) M PAF-acether. IL-2 production was brought up to 90% of control values when both PAF-acether and BN 52021 (10(-4) M) were added together to the lymphocyte cultures. These studies suggest a significant immunoregulatory role for PAF-acether and a potential use of BN 52021 as a biological response modifier.     

低氧暴露对大鼠外周血T淋巴细胞活化的影响

目的：探讨低氧暴露后大鼠外周血T淋巴细胞亚群及活化共刺激分子的变化,为干预措施的研究提供科学依据。方法：采用三色免疫荧光标记流式细胞仪分析法,观察在低氧暴露前和模拟海拔8 000 m低氧暴露8 h、3d6、d和10 d后,大鼠外周血T淋巴细胞亚群和T淋巴细胞活化共刺激分子的变化。结果：模拟海拔8 000 m低氧暴露8 h后,大鼠CD3＋、CD8＋、CD8＋CD28-细胞数较低氧前均显著降低（P均〈0.01）;低氧暴露3 d后,除上述变化外,CD4＋CD28＋细胞数也显著降低（P〈0.01）,CD4＋CD28-细胞数显著增加（P〈0.01）;低氧暴露6 d和10 d后,CD3＋、CD4＋呈现进一步降低趋势,CD8＋CD28＋细胞数显著增加（P〈0.01）。结论：说明模拟海拔8 000 m低氧暴露8 h和3 d后,大鼠外周血CD8＋、CD4＋T淋巴细胞活化水平均显著下降,随着低氧暴露时间的延长,CD8＋T淋巴细胞活化水平由降低变为增加。