Cyclosporin receptor on mouse lymphocytes.

Specific binding of [3H]-cyclosporin C (3H-CS-C), an immunosuppressive oligopeptide, was characterized on different mouse lymphocytes. The binding was saturable, time-dependent and reversible; and KD of 1.5 x 10(-7) M and maximum binding capacity Bmax of 35 pmol/4 x 10(6) cells was found for thymocytes. A computerized analysis of the data confirmed a single population of high affinity binding sites. Lymphocytes of different organs differed in their Bmax: thymus greater than spleen greater than mesenteric lymph node (pure T cells) greater than mesenteric lymph node (pure B cells). Erythrocytes did not show a specific binding.     

Cyclosporin A inhibits helper/inducer surface antigen expression on activated human lymphocytes

Helper/inducer (H/I) and cytotoxic/suppressor (C/S) subpopulations of human peripheral blood mononuclear lymphocytes (PBL) were cultured with Concanavalin A (Con A) and Cyclosporin A (CsA) to determine effects on proliferation and surface marker expression. After 72 h in culture, CsA strongly inhibited Con A induced proliferation of H/I and C/S subpopulations in a manner similar to that observed for whole PBL. By flow cytometric analysis, enhanced expression of both the Leu-2a (C/S) and Leu-3a (H/I) surface markers was observed on PBL from con A treated cultures. Addition of CsA to these cultures inhibited the enhanced expression of Leu-3a more than that of Leu-2a. Studies showed that this CsA inhibition of enhanced Leu-3 antigen expression occurred in a concentration dependent manner, was statistically significant throughout a 4 day culture period and did not require addition of C/S cells to mediate the effect. The data suggest that CsA blocks Con A induced blastogenesis at an early stage of activation.     

The effect of acetylsalicylic acid on the metabolism and transformability of human lymphocytes in vitro

Summary Acetylsalicylic acid inhibits the hexokinase activity of human lymphocytes in vitro. The effect is proportional to the concentration and is readily demonstrable at concentrations greater than 2–3 mMols per liter. In agreement with this, a reduction in the glucose-C¹⁴-decarboxylation rate is seen.This inhibitory effect could be the cause of the observed deterioration of the lymphocyte transformation and C¹⁴-thymidine incorporation in the presence of acetylsalicylic acid. Corresponding to the evident glucose-competitive mechanism of the acetylsalicylates, the inhibition of the thymidine incorporation in stimulated lymphocytes was reversed by increasing the glucose concentration of the culture medium. The results would seem to indicate an immunosuppressive action of acetylsalicylic acid.This investigation was supported by a grant from the Deutsche Forschungsgemeinschaft, Bad Godesberg.     

Cyclosporin A inhibits mitogen-activated but not phorbol ester-activated locomotion of human lymphocytes.

Human blood lymphocytes acquire locomotor capacity during 24 hr culture with mitogens such as monoclonal anti-CD3 antibodies, phytohaemagglutinin (PHA), or Staphylococcus aureus Cowan strain (SAC). Activation of locomotor capacity by these agents is blocked by the presence of cyclosporin A (greater than or equal to 10 ng per ml), as measured by inhibition of the development of morphological polarization of the cells in culture and inhibition of their capacity to invade collagen gels. The response to OKT3 is inhibited by lower doses of cyclosporin than the responses to SAC or PHA. Phorbol myristate acetate (PMA) induces locomotion of lymphocytes in culture that is not inhibited by cyclosporin. Cyclosporin has no effect on the locomotion of lymphocytes that already possess locomotor capacity. Thus it neither inhibits immediate stimulus-induced polarization of lymphocytes direct from blood, nor reverses polarization of lymphocytes that have acquired motility in culture. These results suggest that cyclosporin prevents events, occurring during the G1 phase of growth, that are necessary for non-motile lymphocytes to acquire locomotor capacity, but has no effect on the locomotor mechanism itself in already motile cells.     

The inhibitory effect of theophylline on cell cycle kinetics of human lymphocytes in vitro

The effect of theophylline (TF) on proliferation of lymphocytes in phytohaemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) freshly stimulated 3-4 days cultures or of 14-days cultured lymphoblasts was studied. Proliferation was estimated by measurement of incorporation of tritiated thymidine (3H-TdR), and by microscopic analysis of cellular divisions progression up to the 4th generation of cells cultured in presence of 5-bromodeoxyuridine (BrdUrd). The strongest inhibition of proliferation was observed when TF was added at the beginning of the cultures. However, when TF was added for the last 20 h of the cultures, a significant decrease in number of the third (M3) and fourth (M4) generation cells was noted and M3/M4 ratio increased from 6.4 up to 19.1. TF inhibited also incorporation of 3H-TdR into cultured lymphoblasts. The obtained data indicate an inhibitory effect of TF both an activation and cell cycle progression of lymphocytes.     

Enzymes of purine nucleotide metabolism in human lymphocytes

A methodology is presented for systemic analysis of purine enzymes in small lymphocyte subfractions. For the determination of 7 different enzymes of purine metabolism *hypoxanthine-guanine phosphoribosyltransferase (HG-PRT), adenine phosphoribosyltransferase (A-PRT), adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), adenosine kinase (AK), 5'-nucleotidase (5'N), and AMP-deaminase) less than 200,000 peripheral blood lymphocytes are needed. 1000-6000 lyophilised lymphocytes are incubated in micro-incubation vessels (3 microliter) with radioactive substrates for 15-180 min. Separation of substrates and products is achieved by thin-layer chromatography on PEI-cellulose. Addition of BSA to the incubation mixtures results in higher specific enzyme activities and narrower ranges of mean values of a control group.     

Tetrandrine and transmembrane signal transduction: effect on phosphoinositide metabolism, calcium flux and protein kinase C translocation in human lymphocytes

Time-course and dose-dependent studies showed consistent suppression of phosphoinositide turnover in Con A-stimulated human lymphocytes in the presence of the plant alkaloid, tetrandrine. Significant inhibition of Con A-stimulated calcium flux was also observed. Furthermore, protein kinase C activity was also significantly inhibited by tetrandrine irrespective of whether Con A or phorbol myristate acetate was the stimulant. These results suggest that the immunosuppressive properties of tetrandrine are in part mediated by the capacity of tetrandrine to interfere with transmembrane signalling.     

T lineage progenitors: the earliest steps en route to T lymphocytes

T lymphocyte development in the thymus is a tightly regulated stepwise process. The identification and characterization of the earliest T lineage progenitors and their downstream progeny now enables the study of important cellular and molecular mechanisms that control and regulate T lineage commitment and differentiation. Significant progress has been made recently on the developmental relationships of the various cells with T cell progenitor activity identified in mouse bone marrow, blood and thymus, and on the molecular regulation of progenitor homing to the thymus. The essential role of Notch-1 signalling in intrathymic T lineage commitment and subsequent T cell development has been clearly documented.     

D-penicillamine: Mitogenic effect on mouse,rat and human spleen lymphocytes

The effect of D-penicillamine (D-Pen) on the proliferation of cultures of normal mouse, rat, and human spleen lymphocytes and peripheral blood lymphocytes was examined. D-Pen in concentrations of 2×10^–3 M to 8×10^–3 M in serum-free and in serum-containing medium resulted in a highly significant incorporation of³H-TdR by normal mouse and rat spleen cells. Enhanced incorporation of³H-TdR by normal human spleen cells only occurred in serum-containing medium. D-Pen in concentrations of 10^–4 M to 10^–3 M in serum-free and serum-containing medium resulted in significant inhibition of³H-TdR incorporation by normal and mitogen-stimulated mouse and rat spleen cells. Doses of D-Pen greater than 2×10^–2 M strongly inhibited³H-TdR incorporation by both normal and mitogen-stimulated mouse, rat, and human spleen cells and peripheral blood cells. The latter cells were not stimulated or inhibited at lower concentrations of D-Pen.Results from cell depletion and enriching procedures (specific antibody + C' cell killing, employment of athymic, nude spleen cells, adherent and phagocytic cell removal, E rosette cell separation procedures) suggested that target cells in the mouse spleen for D-Pen activation are non-adherent B cells whereas the D-Pen responsive cells in the human spleen probably are T cells.     

Polypeptides on human B lymphocytes associated with cell activation

Two monoclonal antibodies (MoAbs), designated BB-1 and LB-2, react with distinct polypeptides expressed on activated human B cells. The BB-1 MoAb reacted with a 37,000-dalton polypeptide (Bp37) restricted to pre-B and B-cell blasts and B-cell malignancies. The LB-2 MoAb reacted with a 76,000-dalton polypeptide (p76) found on resting B cells but at higher levels on activated B cells and T cells. Buoyant tonsillar lymphoid cells with a germinal center phenotype express higher levels of Bp37 and p76 than do dense B cells of the mantle zone. Furthermore, the expression of Bp37 and p76 on tonsillar B-cell subsets was distinct from other B-cell antigens such as Bp39, Bp95, Bp135, the C3d receptor and surface IgM. Based on biochemical, cross-blocking, and tissue distribution analyses, these antigens appear to be distinct from previously described B cell and B-cell-blast markers.     

Inhibitory effect of somatostatin on human T lymphocytes proliferation

Somatostatin (SOM) was originally described as a growth hormone release inhibiting factor, but SOM and its specific receptors (SOM-r) have been shown to be expressed on both normal and activated T and B lymphocytes and other immunocompetent cells. In the present study we have demonstrated that SOM strongly inhibits the proliferation of human T lymphocytes when stimulated by PHA, Con A or alloantigens. However, SOM was most effective when the T cells were stimulated by an alloantigen rather than a polyclonal activator such as PHA and ConA. Moreover, SOM strongly inhibited the expression of activation markers such as CD69 and CD25 that are expressed on T lymphocytes during alloantigen stimulation. SOM also inhibited both CD28 and CD2 mediated T cell proliferation. Whereas proliferation of T cells induced by the engagement of CD3 antigen using specific mAbs was only marginally affected. Our results would support the concept that in humans SOM plays a key role in the modulation of T cell activation by interfering with the antigen-independent pathways CD2 and CD28.     

Immunomodulators and enzymes of purine metabolism in human lymphocytes

The enzymes ADA and PNP were evaluated in lymphocytic subpopulations in peripheral blood obtained from healthy subjects, elderly subjects and patients with immunoproliferative diseases. Some similar assessments were performed on lymphoid cells from cord blood. Preliminary studies indicate that Thymostimulin can in some cases correct enzymic defects.     

Cyclosporin A inhibition of macrophage colony-stimulating factor (M-CSF) production by activated human T lymphocytes

M-CSF is a pleiotropic cytokine involved in the survival, proliferation, and differentiation of cells of the monocyte/macrophage lineage. M-CSF is produced by numerous cells including CD3-activated T cells. M-CSF serum levels are increased during acute graft rejection. We tested the in vitro production of M-CSF, GM-CSF, IL-2, and IL-4 by T-cell clones costimulated by CD3 and accessory activation pathways and the effects of cyclosporin A and methylprednisolone. The nine clones studied and CD4+ cells purified from peripheral blood mononuclear cells (PBMC) spontaneously produced low levels of M-CSF, which PMA and CD3 mAb strongly enhanced. In contrast to IL-2, CD28 mAb did not further enhance this production. CsA inhibited M-CSF production by clones and purified CD4 T cells. Addition of IL-2, anti IL-2, or anti CD25 mAb to the cultures demonstrated that CsA down-regulated M-CSF synthesis by activated T cells through its inhibition of IL-2 synthesis. These results could help to better understand the complex mechanisms of acute graft rejection and immunosuppression.     

Epstein-Barr virus-induced transformation of human B lymphocytes: the effect of L-leucyl-L-leucine methyl ester on inhibitory T cell populations.

Epstein-Barr virus-mediated transformation of human B lymphocytes is inhibited by human T lymphocytes as well as by interferon-gamma. Removal of the inhibitory cell populations is essential in order to achieve successful transformation in vitro. Cells with the capacity to inhibit outgrowth of lymphoblastoid cell lines can be removed by pretreatment of peripheral blood mononuclear cells with L-leucyl-L-leucine methyl ester. This treatment eliminates monocytes, NK-cells and a CD8+ T cell subpopulation. We now show that such treatment also has toxic effects on other human T cell populations. In addition, CD4+ and/or CD8+ lymphocytes are demonstrated to contain effector cell activities which inhibit outgrowth of EBV-transformed B cells. This inhibitory activity is abolished after treatment of peripheral blood mononuclear cells or purified CD4+ T cells with L-leucyl-L-leucine methyl ester. No evidence was found for a selective toxicity against any subset within the CD4+ or CD8+ T cell populations. However, the capacity of the treated cells, both peripheral blood mononuclear cells and purified CD4+ T lymphocytes, to produce mRNA encoding IFN-gamma, a protein previously shown to downregulate outgrowth of EBV-transformed B cells, was selectively impaired. The results obtained suggest a role for CD4+ T cells to inhibit EBV-induced transformation of B cells.     

Activation of human B lymphocytes VII. the regulatory effect of cyclic adenosine monophosphate on human B cell activation.

Cyclic adenosine monophosphate (cAMP) has been demonstrated to play an integral role in the regulation of B cell activation. By employing a plaque-forming cell (PFC) assay for polyclonal activation of human B lymphocytes, it was demonstrated that dibutyryl cyclic adenosine monophosphate (DB-cAMP) markedly increased the PFC response of pokeweed mitogen (PWM)--stimulated lymphocytes. Inducers of intracellular cAMP effected a comparable enhancement. Co-cultures of fresh lymphocytes with autologous T cells which had been pre-incubated with DB-cAMP produced an enhancement of B cell activation by a selective effect on the T cells. The mechanism of action of this enhancement of the B cell response is most likely a relative increase in helper T cell function resulting from a selective inhibition of suppressor T cells.     

Carbohydrate metabolism in PHA stimulated human peripheral blood lymphocytes.

Human peripheral blood lymphocytes (PBL) stimulated in vitro by phytohemoagglutinin (PHA) manifest augmented glycolysis and oxidation of glucose-1-14C, indicating an increased utilization of the pentose pathway. Lactic acid production, as index of increased glycolysis, follows the same kinetic of thymidine incorporation and can be easily quantitated by an enzymatic assay.     

Polyamine metabolism in prostaglandin E2-treated human T lymphocytes

The effects of Prostaglandin (PG) E2 treatment of human T lymphocytes on polyamine metabolism were investigated. PGE2 is known to inhibit lymphocyte proliferation, while polyamines play an important role in several biochemical processes leading to increased cell growth. Preincubation of T lymphocytes with PGE2 (10^-6 M) for 10 min. was able to increase ornithine decarboxylase (ODC) activity and putrescine as well as spermine levels, while spermidine concentration was drastically reduced. After 30 and 60 min of treatment, a decrease in ODC activity and putrescine concentration was observed. On the contrary, the initial inhibition of sperrnine-NI-acetyl-transferase (SAT) activity was followed by a progressive increase of this catabolic enzyme. These changes were related to modifications of cAMP concentrations. Our data may help clarify the mechanisms underlying the biphasic effect of PGE2, which ultimately leads to inibition of cell proliferation.