Mechanism of histamine-induced inhibition of lymphocyte response to mitogens in mice

Histamine induced, in mice, an inhibition of lymphocyte response to PHA and LPS, at molar concentrations ranging from 10^?3 to 10^?9M. This inhibition occurs as a specific interaction between histamine and T lymphocytes bearing H2-type receptors for this hormone (H + cells) and Ly 2 membrane antigens. Two features of the suppressive activity of this T-cell subpopulation were observed: (i) when histamine is added at the beginning of the culture period with PHA or LPS, it activates the suppressor activity of H + cells which act on the lymphocyte population responding to PHA and LPS; (ii) preincubation of spleen lymphocytes with histamine for 24 hr induces suppressor cells which inhibit the response to PHA, but not to LPS, of syngeneic lymphocytes in a coculture system, and which are radiosensitive. The role of PHA as a second stimulus of histamine-induced suppressor cells, and the relation between these cells and PHA or Con A-induced suppressor cells, are discussed.     

Interaction of Bacillus Calmette--Guérin-activated macrophages and neoplastic cells in vitro II. The relationship of selective binding to cytolysis

5′-Methylthioadenosine (MTA), a degradation product of S-adenosylmethionine, inhibits DNA and protein synthesis as well as cellular proliferation in human lymphocyte cultures stimulated with mitogens, antigens, or allogeneic cells. MTA (10^?3M) inhibited [³H]Tdy uptake in PHA- or Con A-induced transformation greater than 95%, and inhibited the uptake of both [³H]Tdy and [¹⁴C]Leu to the same degree in lymphocytes stimulated with PPD or allogeneic lymphocytes. MTA inhibition was dose dependent, inhibition being lost when exogenous levels reached approximately 10^?5M. The inhibitory effects of MTA were not produced by cytotoxicity since MTA-inhibited cells washed free of this compound could be stimulated at least as well as uninhibited cells. Understanding the mode of action of MTA and the mechanisms controlling its metabolism may lead to new approaches for regulating cellular proliferation.     

Augmentation of proliferation and generation of specific cytotoxic cells in human mixed lymphocyte culture reactions by colchicine

Cellular proliferation and generation of cytotoxic lymphocytes in mixed lymphocyte culture reactions (MLC) results from cellular interactions initiated by individual differences in cell surface structures determined by the HLA-D locus. Cellular microtubular assemblies (MTA) modulate cell shape and surface architecture and have also been implicated in mediating stimulatory signals from the lymphocyte plasma membrane to intracellular sites. To assess the role of MTA in alloactivation, we tested the effect of colchicine, a microtubule-disrupting alkaloid, on the MLC. Diametrically opposite results were observed depending upon the colchicine treatment protocol. Brief exposure of stimulating cells to 10^?6M colchicine resulted in an increase in [³H]thymidine incorporation from 30, 694 ± 2787 to 47,345 ± 4361 cpm/culture (mean ± SEM) (P < 0.001), exposure of responding cells to 10^?6M colchicine resulted in an increase from 33,054 ± 4012 to 46,790 ± 5458 cpm/culture (P < 0.01), and exposure of both cells to 10^?6M colchicine resulted in an increase from 33,054 ± 4012 to 52,685 ± 6720 cpm/culture (P < 0.01). However, direct addition of colchicine to a final concentration of 10^?6M to MLCs at different times resulted in complete suppression of proliferation when added as late as 96 hr, and 63% suppression when added at 120 hr. Pretreatment of stimulating, responding, or both cells with lumicolchicine did not enhance proliferation. Pretreatment of cells with 10^?6 and 10^?4 M colchicine enhanced proliferation, while pretreatment with 10^?2M colchicine prevented blastogenesis. The potentiation of proliferation induced by colchicine was evident as early as 48 hr after the initiation of the MLC. Generation of specific cytotoxic cells in the MLC was also enhanced by exposing lymphocytes to colchicine prior to the proliferative phase in six out of eight experiments (specific chromium release = 168% of control) despite constant effector:target ratios. These findings indicate that early disruption of microtubules leads to enhanced cellular proliferation and generation of cytotoxic lymphocytes in allogeneic one-way MLCs and suggest that the state of polymerization of the MTA may modulate immune responses involving cell-cell interactions.     

Chicken Harder's gland: evidence for a relatively pure bursa-dependent lymphoid cell population

Theophylline, caffeine, and dibutyryl cAMP, agents that elevate intracellular levels of cyclic AMP, were found to inhibit cytotoxin elaboration by PHA-stimulated human lymphocytes. Cytotoxins in diluted supernatants from 3-day lymphocyte cultures were assayed by ⁵¹Cr release from L cells. Addition of the agents to the lymphocyte cultures inhibited cytotoxin elaboration 70–90% at 10^?3M and 20–50% at 10^?5M. In addition, it was found that the inhibition is reversible and occurs at a step other than the initial mitogen triggering.The inhibition of cytotoxin elaboration by these agents correlates strikingly with their inhibition of lymphocyte-mediated target cell lysis. The results are consistent with the hypothesis that cytotoxin is the mediator of target cell killing by lymphocytes.     

Regulation of immune responses. I. Effects of cyclic AMP and cyclic GMP on immune induction.

Agents that raise intracellular cAMP levels (dibutyryl cyclic AMP, aminophylline, adenosine and butyric acid) increase the magnitude of an in vitro primary humoral immune response when added at 10^?3M during the first 12 hr of a 108 hr culture. Under the same conditions, cGMP has no direct effect but inhibits cAMP-mediated stimulation. DbcAMP (10^?3M or 10^?4M), present from 0 to 12 hr, also increases the number of cytotoxic lymphocytes in CBA/J (H-2^k) spleen cell cultures stimulated in a one-way mixed lymphocyte reaction with DBA/2J (H-2^d) spleen cells. The dbcAMP effect is antigen-dependent in both humoral and cell-mediated immunity and antigen-specific in the case of humoral responses.     

Histamine suppression of human lymphocyte responses to mitogens.

Exogenously added histamine in non-cytotoxic concentrations (10^?5?10^?3M) suppresses in vitro proliferation of lymphocytes induced by PHA or Concanavalin A. This suppressive effect was observed when histamine was present for as short as $\text{1}\text{2}$ hr in the beginning of the culture. Histamine, in concentrations as high as 10^?3M, did not cause increased release of isotope from ⁵¹Cr-labeled lymphocytes following 4 hr of incubation. The histamine H₂ receptor antagonist, metiamide, but not the H₁ receptor antagonists diphenhydramine or chlorpheniramine, blocked the histamine suppressive effect. Some of the biological implications of these findings are discussed.     

Antibody-dependent lymphocyte mediated cytotoxicity mechanism and modulation by cyclic nucleotides.

The injury to antibody coated thymocytes by the “K cell” among nonsensitized rat splenocytes was modulated by altering the cellular level of cAMP and cGMP. Preincubation of the attacking cell population with 1 μg/ml cholera toxin caused an elevation of cAMP levels of 1–18 pM per 10⁷ cells and was associated with a proportionate reduction in cytotoxicity. Agents which are known to raise cAMP levels including the Prostaglandins (PG) E₁ (10^?7–10^?5 M), PGF_2α (10^?5–10^?3 M), PGA₁ (10^?9–10^?5M), and theophylline (10^?3 M) also produced marked suppression of antibody dependent lymphocyte mediated cytotoxicity. Direct elevation of cellular levels of cGMP by the analog 8-bromo cGMP (5 × 10^?6M) led to augmentation of cytotoxicity. Removal by EDTA and MgEDTA of calcium and magnesium ions from the culture media markedly inhibited cytotoxicity.     

The effect of levamisole on lymphocyte protein synthesis in vitro.

Human peripheral blood lymphocytes from normal donors were studied to determine the effect of levamisole, an immunostimulating agent, on lymphocyte protein synthesis in vitro. At a concentration of 7.5 × 10^?6M, levamisole stimulation resulted in a maximal increase in protein synthesis of 136% of control. Levamisole caused no increase in protein synthesis of “T-cell depleted” populations but increased protein synthesis of “T-cell enriched” populations to a degree greater than that observed with whole lymphocyte populations. These results demonstrate that levamisole increases lymphocyte protein synthesis and suggest that the increase is due to selective stimulation of T-cells. These results provide an explanation for the effects of levamisole on lymphokine production and E-rosette formation previously reported.     

Prostaglandin-mediated regulation of the mixed lymphocyte culture and generation of cytotoxic cells

We examined the role of prostaglandins or prostaglandin-producing cells in the regulation of proliferation and generation of specific cytotoxicity in one-way mixed lymphocyte cultures of mouse spleen cells. Cultures treated with indomethacin or other prostaglandin synthesis inhibitors resulted in enhanced proliferation and cytotoxicity. The level of prostaglandins produced in vitro, as measured by RIA, was 10^?8M and was found to be completely blocked by indomethacin. Adding back 10^?8M PG restored baseline (control) proliferative responses. Kinetics of the enhanced MLC response were unchanged from controls as were the specificities of the cytotoxic cells. Cells from indomethacin-treated cultures were more efficient at killing targets than those from control cultures. Prostaglandins appear to have a preferential effect on the induction of cytotoxic cells.     

Endogenous prostaglandin E2 enhances polyclonal immunoglobulin production by tonically inhibiting T suppressor cell activity

The immunoregulatory effect of endogenous prostaglandin E₂ (PGE₂) on immunoglobulin production was studied in an in vitro culture system of human peripheral blood mononuclear cells, stimulated with pokeweed mitogen (PWM). Three different cyclooxygenase inhibitors (indomethacin, carprofen, and piroxicam) suppressed Ig synthesis by ~50%. This inhibitory effect could be reversed by adding low doses of exogenous PGE₂ (3 × 10^?9 to 3 × 10^?8M). These doses are endogenously produced in PWM-stimulated cultures, and a concentration of 2 × 10^?8M is reached after 48 hr of culture. When B cells were directly stimulated with helper factor, PGE₂ did not enhance Ig production and doses of 3 × 10^?7M to 3 × 10^?6M were inhibitory. The effects of indomethacin and PGE₂ were eliminated when T cells were irradiated or treated with mitomycin prior to culture. The enhancing effects of PGE₂ were substantially reduced after OKT8(+) T cells were removed from the system. PWM-stimulated cultures of lymphocytes from healthy subjects over age 70 were more sensitive to inhibition by indomethacin and to stimulation by PGE₂ than were cultures of lymphocytes from young controls. Thus the major role of endogenous PGE in polyclonal Ig production in vitro is to tonically inhibit a radiosensitive, OKT8(+) suppressor T cell. This tonic inhibition is increased in subjects over 70, which provides one explanation for decreased suppressor cell function in elderly subjects.     

Macrophage-like suppressor cells in rats. II. Evidence for a quantitative rather than a qualitative change in tumour-bearer animals

In order to investigate the mechanisms of steroid-induced inhibition of concanavalin A stimulation, we have compared, in mouse lymphoid cells, the ability of various steroids to block transformation with their affinity for glucocorticoid receptors. Our results suggest several possible explanations for the inhibitory effects of the various steroids tested. The action of glucocorticoids, which are inhibitors at concentrations within the physiological range (10^?8–10^?7M) is likely to be mediated through an interaction with specific cytosolic binding sites leading to an overall inhibition of cell metabolism. In contrast, sex steroids only inhibit at pharmacological concentrations, equal to or higher than 10^?5M. These compounds, which do not bind to glucocorticoid receptors, probably act in a “nonspecific” manner at the level of the cell membrane. The effect of 25-OH-cholesterol, a selective inhibitor of cholesterol synthesis, suggests that the level of sterol formation controls in part the proliferative activity of the cells.     

Growth inhibition of mouse lymphoma cells, L5178Y, in vitro, by glucocorticoids : Alterations in choline metabolism

The effects of prednisolone and other glucocorticoids were studied in a cell culture system of L5178Y mouse lymphoma. A relatively high level of 8 × 10⁻⁶ M prednisolone was required to reduce growth by 50%. The response was due to an increase in generation time and was not a result of the cytolytic effects of the steroid. A synchronous cell population was obtained by successive treatment with excess thymidine and Colcemid. Using this technic it was determined that an exposure to prednisolone for a time longer than a complete cell cycle was required in order to bring about inhibition of cell multiplication. In the course of experiments involving brief exposure to prednisolone, it was determined that prednisolone when added to synchronized cells inhibited incorporation of ¹⁴C-choline into the lipid cell fraction. The inhibition of choline incorporation in cells selected for increased resistance to prednisolone was about half that observed in steroid-sensitive cells.     

Differential effects of ouabain on human cell-mediated cytotoxicity. II. Stimulation of monocyte-mediated, spontaneous cytotoxicity against chicken red cell targets.

We have previously shown that ouabain inhibits mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) against chicken red cell (CRC) targets. We now report that ouabain increases spontaneous killing of CRC targets in the absence of mitogen or antibody. Spontaneous cytotoxicity by fresh mononuclear leukocytes (MNL) was enhanced by ouabain in a dose-dependent fashion and was maximal at a ouabain concentration of 5 × 10^?5M. Removal of phagocytic cells from the MNL effector cell population abrogated ouabain-induced spontaneous cytotoxicity, suggesting that the effector cell activated by ouabain was a monocyte. Ouabain-induced spontaneous cytotoxicity was relatively inefficient compared to MICC or ADCC and was only demonstrated consistently at effector:target cell ratios higher than those routinely employed for MICC and ADCC. Very low concentrations of ouabain (5 × 10^?9M) also enhanced spontaneous cytotoxicity of MNL precultured for 7 days, when added at either Day 0 or Day 6 of preculture. The cell effecting spontaneous cytotoxicity after 7 days of culture has been previously shown to be a monocyte. Thus, ouabain has opposing effects on cell-mediated cytotoxic functions: it inhibits MICC and ADCC against CRC targets, but stimulates spontaneous, monocyte-mediated cytotoxicity against the same targets.     

Adenosine deaminase and immunodeficiency: An in vitro model

We have examined the effect of adenosine and EHNA, a competitive inhibitor of adenosine deaminase (ADA), upon the ability of human peripheral blood lymphocytes to respond to mitogen. Addition of adenosine at concentrations greater than 10 μm (10^?5m) resulted in inhibition of lymphocyte proliferation at 48 hr of culture, provided that the culture medium was relatively free of ADA activity. The actual concentrations of adenosine remaining in inhibited cultures at the time of harvest were considerably lower than those added initially. EHNA alone also inhibited PHA response (and to a lesser extent PWM and Con A responses), but only at high concentrations. Noninhibitory concentrations of EHNA and adenosine together acted synergistically to produce profound inhibition of lymphocyte proliferation. This may provide an in vitro model to explore further the mechanism of the immunodeficiency associated with deficiency of ADA. Adenosine deaminase activity in stimulated cultures did not differ significantly from that found in unstimulated cultures, and the activity per protein or per DNA actually decreased in stimulated versus unstimulated cultures.     

The mixed lymphocyte reaction: selective activation and inactivation of the stimulating cells.

Allogeneic cells pretreated for 48 hr with 2 × 10^?6M ouabain have lost the capacity to show the mixed lymphocyte reaction (MLR). Analysis of various combinations of cells in the one-way MLR revealed that this effect was on the stimulating cells and not on the responding cells. Pretreatment of cells from both donors with 10^?7M ouabain caused no change in incorporation of labeled thymidine into DNA during the first 5 days of mixed lymphocyte culture; thereafter, as incorporation by the controls declined, that of the pretreated cells continued to increase. This effect was also on the stimulating cells and not on the responders. The irreversible effects of ouabain are thus either to activate or inactivate the stimulating cells depending on the concentration of the drug; there is little or no effect on the responding cells.     

The type and time of occurrence of aminopterin-induced chromosome aberrations in cultured Potorous cells

Many inhibitors of DNA synthesis have been found to induce chromosome aberrations. Our kinetic studies indicate that treatment of cellswith 10^?7M aminopterin in the presence of 10^?4M glycine, 10^?4M hypoxanthine, and 10^?4M thymidine allows continued normal cell growth. Omission of thymidine, a treatment which is known to inhibit DNA synthesis while allowing RNA and protein synthesis to continue, leads to cessation of cell growth. Treament of Potorous cell cultures with aminopterin in the presence of hypoxanthine and glycine without thymidine led to the following observations: (1) only non-exchange chromatid aberrations were formed after aminopterin treatment; (2) the aberrations were induced only in cells treated during S, and the breaks were associated with the replicating region of the chromosome; (3) breaks were observed at the first metaphase after the beginning of treatment; and (4) thymidine could reverse the chromosome-breaking action of aminopterin. A model for the molecular mechanism is suggested.     

Further studies of the selective inhibition by ouabain of antigen-induced proliferation of human lymphocytes

Cultures of human lymphocytes incubated for 48 hr in the presence of 2 × 10^?7M solutions of the cardiotonic steroid ouabain lose the proliferative response to antigens (SL-0, SK-SD) but can still proliferate when stimulated by nonspecific mitogens (PHA, Con A, pokeweed mitogen). The two-way mixed lymphocyte reaction was also irreversibly lost if cells of both donors were subjected to ouabain pretreatment. Neither cell counts nor cell viability (determined by dye exclusion) were significantly affected by the ouabain treatment. Pretreatment of a suspension of macrophages with the cardiac glycoside did not diminish their capacity to restore the proliferative response to antigen of macrophage-depleted lymphocyte suspensions; on the other hand, untreated macrophages could not restore the proliferative response of cultures of ouabain-pretreated lymphocytes. The ouabain treatment did not change the proportion of cells able to bind fluorescent anti-immunoglobulin nor did it modify the proportion of lymphocytes forming rosettes with either untreated, or antibody coated, red cells. Increased concentration of K⁺ in the medium, either during or after the ouabain treatment, did not reduce the ouabain effect. We conclude that the selective loss of certain lymphocyte functions caused by ouabain pretreatment was due to an effect on the lymphocyte and not on the macrophage; the effect was not due to the elimination of a relatively large fraction of the cells nor to a generalized disappearance of membrane antigens and receptors.     

Properties of histamine-induced suppressor factor in the regulation of lymphocyte response to PHA in mice

Splenic T lymphocytes release a suppressor factor into the culture supernatant when incubated for 24 hr with histamine (10^?4M). Histamine-induced suppressor factor (HISF) inhibits lymphocyte response to PHA; it is released by T lymphocytes (either nylon-nonadherent or nylon-adherent lymphocytes) and not by B-cells or macrophages; its production is not observed after depletion of histamine receptor-bearing lymphocytes and is blocked by the H2 receptor antagonist (cimetidine) but not by the H1 receptor antagonist (diphenhydramine). Gel filtration by Sephadex G100 chromatography indicates that HISF had an approximate MW of 45,000 to 68,000. Its inhibitory activity was removed by passage over a histamine RSA-Sepharose column, but not by passage over rabbit anti-mouse Ig-Sepharose column; it was blocked by prostaglandin synthetase inhibitor (PGS) (Ro 205720) indicating that this activity is mediated by a prostaglandin (PG) synthesis.     

Cytokinin Reversal of Abscisic Acid Inhibition of Growth and α-Amylase Synthesis in Barley Seed

The effect of cytokinin, kinetin, on abscisic acid (dormin) inhibition of α-amylase synthesis and growth in intact barley seed was investigated. Abscisic acid at 5 × 10^?5M nearly completely inhibited growth response and α-amylase synthesis in barley seed. Kinetin reversed to a large extent abscisic acid inhibition of α-aniylase synthesis and coleoptile growth. The response curves of α-amylase synthesis and coleoptile growth in presence of a fixed amount of abscisic acid (6 × l0^?6M) and increasing concentrations of kinetin (from 5 × l0^?7M to 5 × 10^?5 M) showed remarkable similarity. Kinetin and abscisic acid caused synergistic inhibition of root growth. Gibberellic acid was far less effective than kinetin in reversing abscisic acid inhibition of α-amylase synthesis and coleoptile growth. A combination of kinetin and gibberellic acid caused nearly complete reversal of abscisic acid inhibition of α-amylase synthesis but not the abscisic acid inhibition of growth. The results suggest that factors controlling α-amylase synthesis may not have a dominant role in all growth responses of the seed. Kinetin possibly acts by removing the abscisic acid inhibition of enzyme specific sites thereby allowing gibberellic acid to function to produce α-amylase.     

Moniezia expansa: Subcellular distribution,kinetic properties of acetylcholinesterase and effects of inhibitors and anthelmintics

Acetylcholinesterase (EC 3:1:1:7) has been demonstrated biochemically within partially purified whole worm homogenates of Moniezia expansa. Linear activity occurred with temperature, enzyme concentration, and time. The pH optimum was 8.5 and the Michaelis constant 2.8 mM with inhibition by excess substrate. Inhibitor and specific substrate studies indicated that butyrylcholinesterase was probably absent. The molecular weight of AChE was in excess of 300,000. Greatest activity occurred in the 22,000 and 100,000g particulate fractions. Ultrastructural staining showed that activity was restricted to the ribosomes and cisternae of the rough endoplasmic reticulum. Quinacrine hydrochloride caused 48% inhibition of AChE at 10^?3M and haloxon (di(2-chloroethyl)-3-chloro-4-methyl-7-coumarinyl phosphate) caused 97% inhibition at 10^?4M. No appreciable inhibition (< 25%) occurred with 10^?4M bunamidine hydroxynaphthoate, bephenium hydroxynaphthoate, pyrantel tartrate, p-toluoyl phenyl hydrazone, dichlorphen, thiabendazole, mebendazole, fenbendazole, cambendazole, albendazole, parbendazole, oxibendazole, oxfendazole, praziquantel, piperazine adipate, arecoline hydrobromide, and sodium acetarsol.