Enhancement of mitogen-induced lymphocyte proliferation after preincubation is due to altered sensitivity to prostaglandins

Twenty-four hour preincubation at 37 degrees enhances the mitogen-induced DNA synthesis of human lymphocytes. PGE1, given simultaneously with Con A at the start of lymphocyte culture, inhibits the DNA synthesis. After 24 h preincubation of cells, PGE1 fails to decrease the DNA synthesis. Similarly, preincubation abolished the effect of indomethacin increasing DNA synthesis of freshly separated lymphocytes. The intracellular cAMP level of human mononuclear leukocytes rapidly decreases during in vitro incubation at 37 degrees C. PGE1 elevates the intracellular cAMP of freshly separated lymphocytes to 45 times of its starting level. After 24 h preincubation of cells at 37 degrees C, PGE1 elevates cAMP to a lesser extent. This change of PGE1 action may explain the fact that the effect of exogenous PGE1 and endogenous prostaglandins (the production of which can be inhibited by indomethacin) diminishes after incubation of lymphocytes. It is very likely that the change of the effect of prostaglandins produced in lymphocyte cultures and the spontaneous decrease of intracellular cAMP level explains the enhancement of mitogen-induced lymphocyte proliferation after 24 h preincubation at 37 degrees C.     

Carrageenan-mediated suppression or augmentation of mitogen-induced lymphocyte proliferation

The effects of carrageenan on mitogen-induced lymphocyte proliferation were studied in the guinea pig. According to the dose used, carrageenan displayed opposite effects on thymidine uptake by spleen cells or peripheral blood leukocytes stimulated by Concanavalin A or phytohemagglutinin (25 micrograms ml-1 carrageenan increased whereas 0.25 microgram ml-1 depressed thymidine incorporation). Carrageenan, at the high concentration which increased thymidine uptake by mitogen-stimulated spleen cells, potentiated the enhancing activity of macrophages that was observed with cell suspensions containing 20% macrophages. Conversely, low concentrations of carrageenan abolished the enhancing effect of macrophages. These effects of carrageenan on lymphocyte proliferation could be explained by its activities on a macrophage functional subset rather than on the whole macrophage population.     

Enhancement of lymphocyte proliferation by mouse glandular kallikrein.

Mouse glandular kallikrein (mGK) strongly enhanced the spontaneous and mitogen-induced proliferation of lymphocytes. Both blast formation and 3H-TdR incorporation were dose-dependently enhanced at the same time many cells were killed. The enhancing activity was independent of EGF, because EGF-binding proteins (mGK-9 in mGK-6,9 mixture and mGK-13), renal kallikrein (mGK-6) and human kallikrein all displayed the same enhancement. A serine proteinase inhibitor, diisopropyl fluorophosphate, could block the enhancement by mGK. The new function suggests that mGK is important in the immune system as a regulatory molecule.     

Identification of proliferating lymphocyte subpopulations by combined alkaline phosphatase anti-alkaline phosphatase (APAAP) staining and autoradiography

An improvement in the classification of proliferating ([³H]thymidine incorporating) lymphocyte subpopulations in mitogen- or antigen-stimulated microcultures is described. The binding of subset-specific monoclonal antibodies is detected by the alkaline phosphatase anti-alkaline phosphatase method (APAAP). There are two advantages compared to the peroxidase anti-peroxidase (PAP) method; (1) endogenous enzyme (peroxidase)_activity exhibited by some cells causes no interference, and (2) the red alkaline phosphatase staining obtained with new fuchsin provides a far superior contrast to silver grains than conventional peroxidase staining.     

Identity of PB76 differentiation antigen and lymphocyte alkaline phosphatase

Alkaline phosphatases (APases, EC 3.1.3.1) are ecto-enzymes bound to cell membranes by a phosphatidyl-inositol anchor. We have previously shown that APase is present on activated murine B cells and its expression correlates with the process of B cell differentiation into immunoglobulin secretion. Recently, a monoclonal antibody (mAb), G-5-2, that recognizes a 76-kDa molecule preferentially expressed on the surface of pre-B and plasma cells (PB76) was described. Some features shared by APase and PB76 differentiation antigen suggest that the G-5-2 mAb might be specific for lymphocyte APase. Here, we have analyzed this possibility and found an absolute correlation between PB76 expression in cells and their APase activity. Although PB76 has been described as a B cell-restricted marker, PB76 is also expressed on some T cells, such as the YAC-1 T cell lymphoma, that are known to bear APase. Treatment of YAC-1 cells with phosphatidylinositol-specific phospholipase C resulted in a quantitatively correlated removal of both APase and PB76 antigens. Moreover, we demonstrate that PB76 antigen has APase activity using an enzyme-antigen immunoassay with the G-5-2 mAb. We conclude that PB76 and lymphocyte APase are one and the same antigen.     

Enhancement of lymphocyte proliferation assays by use of serum-free medium.

Fetal bovine serum (FBS)-supplemented cell culture medium has been the standard medium used in assays of murine lymphocyte proliferation. While FBS allows cell growth and viability, it requires screening and contains uncharacterized elements which may yield inconsistent results from batch to batch. It also may include growth factors and immunologically reactive proteins that obscure assays for specific responses because of high background proliferation. Recently, chemically defined serum-free media have become available for human lymphocyte culture. We compared several of these media against FBS-supplemented medium and found that one of them, AIM V (Gibco), produced a marked increase in the ratio of positive to background counts in [3H]thymidine incorporation assays of antigen-specific proliferation. Similar results were obtained when responses to mitogenic lectins were examined. AIM V supported proliferation of lymphocytes in a variety of haplotypes, it supported MHC-specific proliferation in response to soluble antigens in a manner consistent with previous reports, and it supported proliferation in response to alloantigen as displayed in the mixed lymphocyte culture. A high ratio of positive to background counts was consistently observed. The results indicate that this serum-free medium enhances the analytic power of proliferation assays.     

Inhibition of diamine oxidase by antimalarial drugs

The antimalarial drugs amodiaquine, quinacrine and chloroquine inhibit the catabolism of putrescine by the rat. Amodiaquine, the most potent of the three, does so in a dose-dependent fashion. This is attributed to the action in vivo of the drugs on diamine oxidase, an enzyme that is inhibited by them in vitro.     

Inhibition of diamine oxidase activity by heparins

Inflammation Research -     

Inhibition of mitogen-induced human lymphocyte responsiveness by polymixin antibiotics

Polymixin antibiotics, polymixin B and polymixin E (colistin) inhibited the mitogen-induced lymphoproliferative response of human lymphocytes. Inhibition of the lymphocyte response to PHA, PWM and Con A was evident at a low concentration of 1 U/ml of antibiotics. Lymphocytes in which the signals for proliferation had occurred were similarly prevented from proliferating. The effects were not due to cell death (toxicity). Since polymixin concentrations at which inhibition of lymphocyte proliferation was observed are employed in tissue culture medium and are also attained in plasma of patients, the results suggest that the use of the antibiotics in lymphocyte cultures limits lymphocyte responsiveness and that patients receiving polymixin antibiotics may experience a state of immunosuppression.     

Measurement of lymphocyte cytotoxicity by assessing endogenous alkaline phosphatase activity of the target cells

A simple, objective, non-isotopic method for testing lymphocyte cytotoxicity is described. The evaluation of the test is based on photometric measurement of endogenous alkaline phosphatese activity of the residual adherent target cells.     

Effects of alkaline phosphatase inhibitors on intestinal transfer of calcium

Adults rats receive by gavage 10 mM CaCl2 [+45Ca] solution containing or not 100 mM glucid, 10-100 mM EDTA or these both compounds. Calcium transfer is determined by the evaluation of [45Ca] in intestine and feces as well as in plasma and femur. Basic Ca++ transfer which corresponds to the CaCl2 solution was doubled in the presence of glucids. EDTA addition abolishes completely the glucid effect, exercising any influence on basic CA++ transfer. Injected into ligaturated ileal loop, the glucid gives the same effect. But the addition of phosphate not only removed glucid action but also inhibits the basic Ca++ transfer. The glucids, known acceptors of phosphate, increase Ca transfer. EDTA and phosphate, alkaline phosphatase inhibitors, exhibit an opposite effect. Phlorizin, as it was seen previously, acts exactly as EDTA. All these facts question: the simultaneous transfer of Ca and glucid, the possibility of a glucid phosphorylation, the part in these events of alkaline phosphatase, phosphorylating, phosphatasic and phosphorylable microvillar protein.     

Inhibition of diamine oxidase by antihistaminic agents and related drugs

Various drugs were tested as inhibitors of diamine oxidase on the basis of chemical relationships to the enzyme substrates.It was found that serotonine tryptamine and phenformin are good competitive inhibitors while cimetidine and pheniprazine are non-competitive inhibitors. Other antihistaminic drugs like promethazine are less powerful inhibitors.     

Inhibition of plant and mammalian diamine oxidase by substrate analogues

Imidazoles, aliphatic substrate analogues and the natural dipeptides, carnosine and anserine, were investigated as inhibitors of diamine oxidase from the pig kidney, human pregnancy plasma and pea seedlings. Imidazole, methyli-midazoles,N-acetylimidazole, histamine andN -methyl-histamine are relatively potent inhibitors of mammalian diamine oxidase showing no influence on plant enzymes. Anserine and carnosine are inhibitors of pig kidney and pea seedling enzymes.K ₁ values are 2 M and 10 M respectively. Investigated natural derivatives of putrescine and cadaverine have no influence on diamine oxidase of different origin.In conclusion, we present some evidence to suggest that mammalian diamine oxidase, despite a high reaction rate with putrescine, is better adapted to histamine oxidation, whereas for plant enzymes the diamines are preferred substrates.     

Activation of human T lymphocytes I. Requirements for mitogen-induced proliferation of antigen-specific T lymphocyte clones

In a model system for accessory cell (AC)-dependent mitogen-induced T cell proliferation the response of several human antigen-specific, HLA-restricted helper T lymphocyte clones (HTLC) to mitogens was studied. It was found that the HTLC themselves did not or only weakly respond to various mitogens or to oxidation by galactose oxidase, but that the response could be strongly increased if certain tumor cell lines were added to the assay as AC. Pretreatment with lectins or oxidation of either HTLC or AC was effective in stimulating the proliferation of the T cells in this system. Reduction of the aldehydes formed during oxidation completely abolished the stimulatory activity of oxidized B lymphoblastoid cell line. This shows that crosslinking of T cell and AC is required to induce proliferation. When several established cell lines were tested for their capacity to function as AC in this system, profound differences in AC activity were detected. The inability of cells with poor AC activity to stimulate the HTLC was not due to trivial reasons, such as requirements for different mitogen concentrations, a decreased binding of mitogens or suppressive effects. Furthermore, AC activity was not dependent on the presence of Ia antigens on the AC. These findings are discussed with regard to the mechanism of mitogen-induced T cell activation.     

Inhibition of mitogen-induced human lymphocyte proliferative responses by tetracycline analogues.

The effect of tetracyclines on mitogen-induced proliferative responses of human lymphocytes was examined. The results showed that of the three tetracycline analogues studied, doxycycline possessed the most potent inhibiting effects. This occurred at drug concentrations (1--10 micrograms/ml) easily attainable in serum during conventional dosage schedules. Other investigations have shown that tetracyclines also interfere with neutrophil function. Taken together, these findings may have clinical significance. Recovery from serious infections generally requires some minimal host immune responses, and the immunosuppressive side-effects of tetracyclines may have detrimental effects on patients with debilitating illnesses or impaired immunological defence mechanisms. Furthermore, tetracyclines may share some common properties of conventional immunosuppressive drugs, such as cytotoxicity, teratogenicity and cancerogenicity. The long-term use of tetracyclines for conditions such as chronic bronchitis, bronchiectasis and acne vulgaris needs to be re-examined.     

Analysis of mammalian diamine oxidase genes

Inflammation Research -     

Purification of human intestinal diamine oxidase

Inflammation Research -