Transduction of Mitogenic Signals in T Lymphocytes

Treatment of human T lymphocytes with mitogenic ligands, such as concanavalin A (Con A), induces a rapid activation of the enzyme ornithine decarboxylase (ODC). This activation occurs within minutes and is completely inhibited when the cells are treated with 1 mM Li+ (in an inositol-free medium) prior to stimulation with Con A. In the presence of 1 mM myo-inositol Li+ has no effect on the Con A-induced activation of ODC. To elucidate why inositol is needed for the mitogen-induced activation of ODC in T lymphocytes, we tested the ability of different inositol metabolites to reverse the inhibitory effect of Li+. Here we report that inositol phospholipids, in addition to inositol, reverse the Li+-induced inhibition of ODC activation, while all other inositol derivatives tested were ineffective. This indicates that Li+ does not block the activation of ODC by inhibiting the generation of inositol phosphates, but rather by a mechanism which is circumvented if inositol phospholipids are added. The molecular mechanisms involved in the rapid activation of ODC by mitogens in human T lymphocytes apparently require inositol phospholipids, but are not directly mediated by inositol-1,4,5-trisphosphate (IP3) alone, diacylglycerol alone, or other inositol phosphates.     

Lymphocyte activation and immune regulation

Occupation of cell surface receptors on lymphocytes by appropriate antigens and/or growth and differentiation factors initiate activation steps essential for acquisition of differentiated functions and clonal expansion. The diversity of lymphocyte antigen-specific receptors and selective expression of binding sites for distinct growth and differentiation factors form the basis for the specificity of the immune response. In contrast to the diversity of receptor-associated recognition systems, transduction of signals to lymphocyte nuclei seems to occur via a limited number of biochemical pathways. One ubiquitous mechanism of signal transduction involves the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) and formation of two second messengers, diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP₃), which trigger numerous physiological responses in T and B lymphocytes. Lymphocytes which are triggered by different or even identical ligands can use a single signal transduction pathway but respond with any of several genetically predetermined effector functions. Ligands and cell surface receptors that participate in lymphocyte activation, and the intracellular events accompanying this activation step were the topic of a recent meeting.     

Gastrointestinal Regulatory Peptides Modulate Mouse Lymphocyte Functions Under Serum-Free Conditions In Vitro

Conditions are described for performing mitogen (Concanavalin A, Con A; lipopolysaccharide, LPS) and mixed lymphocyte reaction (MLR) cultures using serum-free medium. The effects of exogenously adding several gastrointestinal regulatory peptides (β-endorphin, substance P, met-enkephalin, vasoactive intestinal peptide, bombesin and somatostatin) on the incorporation of ³H-methyl-thymidine was determined. It was observed that mitogen stimulation of lymph node cells with Con A was inhibited (70% of control) by vasoactive intestinal peptide (VIP) but spleen cells stimulated by LPS were insensitive to immunomodulation (98% of control). The ability of VIP to inhibit Con A induced thymidine incorporation was concentration dependent (10^-6 to 10^-18 M) and was not attributable to kinetic shifts or cell toxicity. None of the other tested neuropeptides affected Con A or LPS induced blastogenesis. MLR cultures were inhibited by VIP, β-endorphin and somatostatin in a biphasic manner with maximal inhibition observed at 10^-8 to 10^-12 M. Both substance P and bombesin exhibited slight immunoenhancing properties at 10^-14 to 10^-18 M. Met-enkephalin was ineffective as an immunomodulator of MLR cultures. The utility of using serum-free medium in identifying neuropeptides with immunomodulatory properties are discussed.     

Mrphological and Functional Changes in Mouse Splenic Lymphocytes Following in Vivo and in Vitro Exposure to Chlorphentermine

With repeated administration to animals, the cationic, amphiphilic drug, cnlorphentermine (CP), has been shown by others to induce a phospholipidosis in lymphocytes. In the present study mouse splenic lymphocytes, exposed to CP, either in vivo or in vitro, developed morphological changes consistant With the induction of phospholipidosis. In addition, CP induced functional changes in lymphocytes. Mice, treated with CP in vivo, demonstrated a significantly depressed ability to generate a delayed hypersensitivity response or to produce antibody-secreting cells against de novo antigens. Mouse splenic lymphocytes, exposed to 10^-7 M CP for 3 days in vitro, demonstrated a signficantly depressed blastogenic response to the mitogens phytohemagglutinin, concanavalin A and lipopolysaccharide.

CP inhibited an event that occurred early during lymphocyte activation, but was subsequent to mitogen/receptor coupling. In addition, CP significantly depressed the increased uptake of choline that occurs in lymphocytes following cellular activation. Since the presence of phospholipidosis is indicative of an impairment in phospho lipid metabolism, these results taken together provide evidence for a relationship between this phenomenon and a ltered immune function.     

Cyclosporine does not inhibit mitogen-induced inositol phospholipid degradation in mouse lymphocytes.

The degradation of phosphatidylinositol bisphosphate (PIP2) to diacylglycerol and inositol trisphosphate is elicited by ligand-receptor interactions in many cell types, and may be involved in the induction of cell growth. The mitogens concanavalin A and anti-immunoglobulin antibodies have previously been shown to induce degradation of PIP2 in mouse thymocytes and B cells, respectively. We have now investigated the effects of the immunosuppressive peptide cyclosporine (CS) on this response, since CS appears to inhibit an early step in lymphocyte activation by mitogens that induce PIP2 degradation and Ca2+ mobilization. We found that CS, at doses that completely abrogated the proliferative responses, did not affect the degradation of inositol phospholipids in either thymocytes or B cells. We therefore conclude that if PIP2 degradation is implicated in lymphocyte activation, then CS does not interfere with the second messenger production initiated by PIP2 breakdown, but rather with a later event(s) elicited by this pathway.     

Differential Effect of Mixed D1/D2 and Selective D2 Dopaminergic Antagonists on Mouse T and B Lymphocyte Proliferation and Interleukin Production In Vitro

The effects of some dopaminergic antagonists were investigated on mouse lymphocyte proliferative responses in vitro. The mixed D₁/D₂ dopaminergic antagonists chlorpromazine, haloperidol and fIupentixol inhibited ³H-Thymidine incorporation into adult BALB/c mouse spleen cells stimulated by concanavalin A, lipopolysaccharide from Escherichia coli, and allogenic cells in a mixed lymphocyte reaction. The inhibition was achieved at concentrations greater than 10^-6M. It was not accounted for by decreased cell viability and it was no longer demonstrable when the compound was added 24h or 48h after the mitogenic stimulus. Conversely selective D₂ dopaminergic antagonists sulpiride, metoclopramide and domperidone had no inhibitory effect at concentrations ranging from 10^-9 to 10^-5 or 10^-4M. The three mixed D_l/D₂ antagonists inhibited the mitogenic effect of interleukin-1 on concanavalin A-stimulated thymocytes, but not the activity of interleukin-2 on the proliferaiton of the CTLL-2 cell line. The mixed D_l/D₂ antagonists interfered with the production of interleukin-2 but not with that of interleukin-1. These results indicate that dopaminergic antagonists may differerentially affect lymphocyte proliferative responses to T or B cell mitogens or alloantigens. The mechanisms involved in terms of receptor specific or non specific phenomenons are discussed.     

In vitro analysis of delayed immune response in a bat, Pteropus giganteus: process of con-A mediated activation

In vitro activation of lymphocytes from the bat, Pteropus giganteus with Con A has been analysed in terms of blastogenesis, DNA synthesis and the effector function, the cytotoxic response. We have observed that lymphocytes of P. giganteus are capable of responding to Con A stimulus and the peak of blastogenesis, DNA synthesis and cytotoxic response have been found at 120 hours with the optimal dose of 10 micrograms X ml-1 Con A; it seems the process of activation in the bat's lymphocytes is delayed compared to this in mice. Significance of this delay in the process of activation of immune response in the bat has been discussed.     

Enhancement of Suppressor T Cell Activity by Lymphocyte Promotiong Factor of Bordetella Pertussis

Lymphocyte promoting factor (LPF), a bioproduct of Bordetella pertussis shown previously to exert immunosuppressive effects, was examined for its effect on Concanavalin A (Con A) induced T lymphocyte supressor activity by human lymphocytes. Suppressor cells (5 ± 104) cells/well) first generated with Con A (10 mμg/ml) for 24h were co-cultured with responder cells (5 × 10⁴ cells/ well for another 72h in the presence of Con A (2.5, 5, 20 mμg/ml). In this culture condition, LPF (20 mμg) and/or histamine 10^-5 M or 10^-6 M/well were added for comparison. The suppressor activity was assessed by the reduction of the uptake of radioactivity of [³H] thymidine compared to that of Con A alone. Lymphocytes treated with LPF exhibited increase of suppression by 22 to 41%, while LPF and histamine (10^-5 M) together further increased the suppression by 35 to 63% compared to controls. Serial studies on cell population in the culture indicated OKT8(+) cells proliferated after day 6 in culture. These results indicated LPF exerts selective enhancement of T cell suppessor activity by 72h in culture, prior to cell expansion, and that activity can be further strengthened by sensitizing cells to histamine.     

Influence of chronic ethanol consumption on the inositol phospholipid fatty acid composition of human peripheral blood lymphocytes.

The breakdown of inositol phospholipids is an important event after the binding of antigens to the T-cell antigen receptor. In alcoholics, changes either in early or in late steps of lymphocyte activation have been documented, however no study on the role of phosphoinositide fatty acid composition in signal transduction has been reported. We have analyzed the fatty acid pattern of phosphatidylinositol, phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate from peripheral blood lymphocytes of alcoholic patients and healthy controls, in order to point out the possible compositional differences which could interfere with the signal transmission responsible for the early events in lymphocyte activation. In alcoholics, the arachidonic acid relative molar content in all the inositol phospholipid (PtdIns) fractions derived from lymphocytes was lower than in controls; all PtdIns classes appeared much more saturated than the corresponding fractions from control lymphocytes. The different fatty acid pattern of PtdIns in alcoholic patients could be responsible for an altered second messenger production, above all the production of a modified diacylglycerol which, in turn, could cause a different activation pattern of protein kinase C, with a consequent alteration in cell proliferation. The decrease in arachidonic acid molar content in the phosphoinositides and particularly in the phosphatidylinositol-4,5-bisphosphate fraction of PBL of alcoholic patients could lead to a reduced synthesis of prostanoids of the (n-6) series, and, as a consequence, to an alteration in the mitogenic response of the cells.     

The Effect of Prostaglandin E1 or E2 OH the in Vitro Blastogenic Response of Lymphocytes from Normal and Tumor-Bearing Mice

The in vitro effect of exogenously added prostaglandin (PG) El or E₂ over concentrations ranges of from 1 × 10^--⁴ to 1 × 10-9 M were studied in order to determine their effect on the in vitro lymphocyte proliferation of thymic and splenic T and B cells from normal and tumor-bearing CD₂ F ₁ mice. It was found that PGE₁ generally caused greater inhibition of blastogenesis than did PGE₂ when reacted with splenic lymphocytes from normal mice. Indeed, PGE₂ was found to be stimulatory for both Con A^- and LPS-sensitive normal splenic lymphocytes. Both PGE_l and PGE₂ caused potent inhibition of Con A^- and PHA-sensitive splenic lymphocytes from the tumor-bearing mice. Additionally, PGE₂ was found to stimulate the LPS-reactive lymphocytes from the tumored mice. PGE₁ and PGE₂ both inhibited the Con A- and PHA-reactive thymic lymphocytes from normal mice at the lower concentrations studied, i.e., 10^--⁴ to 10^--⁶ M. Thereafter, at concentration ranoes of from 10^--⁷ to 10^--⁹ M both PGE₁ and PGE₂ were both found to be stimulatory. Finally, both PGE₁ and PGE₂ at all concentrations studied, strongly inhibited the thymic lymphocytes from tumored mice.     

Suppressed peripheral blood lymphocyte blastogenesis in pre- and postpartal sheep by chronic heat-stress, and suppressive property of heat-stressed sheep serum on lymphocytes

Phytohemagglutinin (PHA) and concanavalin A (Con A)-induced blastogenesis of peripheral blood lymphocytes was examined in heat-stressed pre- and postpartal sheep. The peak responses of lymphocytes to PHA and Con A in heat-stressed sheep revealed significant reduction before and after parturition compared with those in the corresponding control animals kept under thermoneutral conditions. Furthermore, the effect of serum from control or heat-stressed sheep on PHA-induced lymphocyte blastogenesis was examined. Supplementation of serum from heat-stressed sheep significantly suppressed the blastogenesis of lymphocytes obtained from healthy sheep, bovine, and human donors. Unlike dexamethasone, heat-stressed sheep serum did not inhibit IL-2 production by PHA-stimulated human peripheral blood lymphocytes. These results indicate that the immunosuppression of heat-stressed sheep is in part mediated by serum factor(s) that can modulate T-cell function in a species nonspecific manner.     

NEUROPEPTIDES MODULATE A MURINE MONOCYTE/MACROPHAGE CELL LINE CAPACITY FOR PHAGOCYTOSIS AND KILLING OF LEISHMANIA MAJOR PARASITES

Host-parasite interactions and their outcome constitute a critical and challenging step in disease establishment in cutaneous leishmaniasis. In the present in vitro study we investigated the possible modulating effects of both sensory and autonomic neuropeptides that normally exist in human and mouse skin, on the uptake and leishmanicidal capacity of macrophages on Leishmania (L.) major parasites, using a monocyte/macrophage murine cell line (Raw 264.7). The sensory neuropeptides somatostatin (SOM), calcitonin gene-related peptide (CGRP) and substance P (SP) suppressed the macrophage capacity for phagocytosing L. major promastigotes at different concentrations, 10^-10-10^-5M, however, the suppressive effect of SP does not reach a significant level. CGRP and SP enhanced the leishmanicidal capacity of macrophages at 10^-7M, and 10^-5 M, respectively, whereas SOM was without effect.

The autonomic neuropeptides vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) both suppressed the phagocytic and leishmanicidal capacities of macrophages at various concentrations, 10^-10-10^-5M.

The findings indicate that neuropeptides have modulating effects on macrophage-L. major interactions. These effects might be exerted by a direct action on macrophages or indirectly through induction of other mediators.     

A Short Incubation with PGE2 Affects the Proliferative Response of T Lymphocytes to PWM

In this study we have investigated the role of PGE2 in the activation of human T lymphocytes by PWM. A preincubation of these cells with molar concentrations of the prostaglandin ranging from 10^-9 M to 10^-4 M is able to reduce the expression of IL-2R and CD71 on T lymphocyte membrane during the first days of culture, while the DR molecule which is expressed later in the same experimental conditions is not affected by the treatment of T lymphocytes with PGE2. The PGE2-induced inhibition of IL-2R and CD71 well correlates with the reduction of ³H-thymidine incorporation by T cells, indicating that a preincubation of T lymphocytes with PGE2 profoundly affects the proliferative apparatus of these cells when they are stimulated by PWM.     

Inhibition of antigen- and lectin-induced proliferation of rat spleen cells by a Taenia taeniaeformis proteinase inhibitor

Rat splenic lymphocytes, cultured in vitro for 3 days in the presence of a larval cestode proteinase inhibitor, exhibited a marked suppression of proliferation when stimulated with Con A, PHA, PWM and ovalbumin. Reduced responsiveness was observed over a full range of concentrations of Con A (16-fold), PHA (50-fold), PWM (four-fold) and ovalbumin (16-fold). These results indicated that the inhibitory action could not be overcome by increasing the mitogen or antigen doses beyond optimal levels. This suppressive effect disappeared when the Taenia taeniaeformis proteinase inhibitor was added 20 h after the initiation of culture, suggesting that the inhibitor affects lymphocyte blastogenesis during the early stages of lymphocyte activation.     

Ontogeny of mitogen responsive lymphocytes in the bovine fetus

Bovine fetal lymphocytes were examined for their ability to respond to the mitogens phytohemagglutinin (PHA), Concanavalin A (ConA) and pokeweed mitogen (PWM) in the lymphocyte blastogenesis test (LBT). PHA-, Con A- and PWM-responsive lymphocytes appeared simultaneously at 75-80 days of gestation. The response increased with age of the fetus until, by 120 days of gestation, the response to PHA, Con A and PWM of many fetuses was in the range of values obtained with lymphocytes from normal adult cattle.     

The effect of heliox treatment in a rat model of focal transient cerebral ischemia

At nerve terminals G protein coupled receptors modulate neurotransmitter release probability. We recently showed that prolonged activation of metabotropic glutamate receptor 7, mGlu7 receptor, potentiates glutamate release. This signalling involves phospholipase C activation via a pertussis toxin insensitive G protein, the hydrolysis of phosphatidylinositol (4,5)-bisphosphate, and the subsequent activation of the non-kinase diacylglycerol binding protein Munc13-1 which primes synaptic vesicle for exocytosis at the active zone. Here we found that inhibitors of diacylglycerol metabolism (diacylglycerol kinase inhibitor II and diacylglycerol lipase inhibitor RHC80267) remarkably reduce the time of mGlu7 receptor stimulation required for glutamate release potentiation in mice cerebrocortical nerve terminals. We conclude that changes in diacylglycerol levels at nerve terminals control the efficiency of the exocytotic release machinery.     

The effects of a new phthalazine derivative (MDL 899) on human lymphocyte functions

MDL-899 is new phthalazine derivative which has been developed as a substitute for hydralazine, which has several undesirable effects, in the treatment of hypertension. The effects of MDL-899 on human lymphocyte functions are analyzed. This drug inhibited in a dose-related fashion the blastogenesis of lymphocytes upon PHA and Con A activation and down-regulated the activation of allogeneic mixed lymphocyte reactions (MLR). On the contrary the drug enhanced the activation of autologous MLR of non T/T type. This effect was five times higher on cells which carried the HLA DR 4 phenotype. The above reported observations suggest that MDL-899, as well as hydralazine, affects the in vitro responsiveness of human lymphocytes mostly in subjects with HLA-DR 4 phenotype. Whether the impact of MDL-899 on immune function gives rise to a lupus like syndrome is not known. For this reason further studies are warranted to assess its long-term in vivo effects.     

Effect of chronic endotoxemia on concanavalin A-stimulated inositol lipid metabolism in rat splenocytes

The effect of a chronic, nonlethal infusion of endotoxin (ET) on the phosphatidylinositol (PI) cycle in rat splenocytes was evaluated. Rats were infused intravenously with sterile saline or Escherichia coli ET (0.1 mg/100 g body weight/24 hr) for 30 hr via subcutaneously implanted osmotic pumps. The splenocytes were then labeled in vitro for 90 min with [32P]PO4 or for 3 hr with [3H]myoinositol to assess the status of the resynthesis and degradative parts of the PI cycle, respectively. PI cycle activity was depressed in splenocytes of ET-infused rats as evidenced by a 25% reduction in the incorporation of [32P]PO4 into phosphatidic acid (PA), PI, and the polyphosphoinositides and by a similar decrease in the production of [3H]inositol phosphates. Stimulation of splenocytes with concanavalin A (Con A) resulted in dose-dependent increases in the incorporation of [32P]PO4 into PA and PI and in the production of [3H]inositol phosphates, indicating that Con A stimulates the PI cycle in these cells. The Con A-stimulated increase in inositol phosphate production was higher in splenocytes from ET-infused rats. We have previously shown that splenocytes from rats infused for 30 hr with ET exhibit a decreased blastogenic responsiveness to Con A and lipopolysaccharide [Spitzer et al., Proc. Soc. Exp. Biol. Med. 186,27, 1987]. The present data do not support the notion that inositol lipid-mediated signaling mechanisms are solely responsible for the expression of the appropriate functional response and suggest that in ET-infused rats there is an uncoupling of the initial response to Con A (i.e., the production of inositol lipid-derived second messengers) and the long-term (i.e., mitogenic) response.     

Chemiluminescence in Human Whole Blood: Modulation by the Cocarcinogens Phorbol Diester and Polychlorobiphenyls

A human whole blood chemiluminescence (CL) assay was established using zymosan as cell activator. Aroclor 1254 was found to inhibit this CL response in a direct linear relation to its concentration, (50% inhibitory dose, (ID₅₀) equal to 5 × 10^-4 M) in diluted blood samples of 10 normal human subjects. In comparison the ID₅₀ of other inhibitors was 1.3 × 10^-3 M for ethylenediamine tetraacetic acid, 3.3 × 10^-3 M for ascorbic acid, 4 × 10^-3 M for reduced glutathione, 1.2 × 10^-3M for ethanol, 2.5 × 10^-1 for methanol and 3.7 × 10^-1 M for dimethyl sulfoxide. Using 12-o-tetradecanoyl-phorbol-13-acetate (TPA) as cell activator the CL response was likewise inhibited by Aroclor 1254 with an ID₅₀ of 4.5 × 10^-4 M. However, it was found that Aroclor 1254 alone has a stimulatory CL effect on otherwise unactivated cells. To compare the mechanisms involved in the CL elicited by the three stimulants zymosan, TPA and Aroclor 1254, the CL signal was measured in the presence of cytochalasin B. Cytochalasin B inhibited zymosan-induced CL, had a smaller inhibitory effect on TPA-induced CL but it could augment the CL response initiated by Aroclor 1254. This pattern of responses implicates Aroclor 1254 in the activation of eicosanoid metabolism as it matches the differential responses reported for arachidonic acid.     

T-Cell Chemiluminescence

The binding of mitogenic lectins phytohaemagglutinin (PHA), concanavalin A (Con A) and/or of monoclonal antibodies to different receptors such as antigen receptor complex or CD2 on human T cells generates increases in the concentrations of inositol triphosphate (IP3) and cytoplasmic free calcium. This T lymphocyte requires the delivery of two signals; the first can be provided by specific monoclonal antibodies or by mitogenic lectins, and the second by a phorbol ester, phorbol myristate acetate (PMA). In other cells such as macrophages, the rise of intracellular calcium via the generation of IP3 and stimulation of protein kinase C can activate the phospholipase A₂, a calcium-dependent enzyme. This enzyme initiates the release of reactive oxygen intermediates and metabolites of arachidonic acid. In order to know whether this other metabolic pathway can be generated in T cells, we tested the capacity of different T-cell lines and clones to produce superoxide anion after stimulation by the above-mentioned activating agents. In this paper, we demonstrate that treatment of the Jurkat human cell line with Con A, PHA, and PMA results in a significant release of reactive oxygen metabolites. Of the various T-cell lines and clones tested, only Jurkat exhibited an oxidative burst. Moreover, none of the antibodies tested (anti-CD3, anti-CD2, and anti-CD28) and known to activate T cells, and none of the immune complexes was able to mediate such an effect. The existence of an oxidative metabolism in at least one T-cell line suggests that T-cell activation may in some instances use another metabolic pathway.