Impairment of TNF-alpha production and action by imidazo[1,2- alpha] quinoxalines, a derivative family which displays potential anti-inflammatory properties.

In a previous study, we analysed the synthesis and properties of a series of imidazo[1,2-alpha]quinoxalines designed in our laboratory as possible imiquimod analogues. We found that these imidazo[1,2-alpha]quinoxalines were in fact potent inhibitors of phosphodiesterase 4 enzymes (PDE4). PDE4 inhibition normally results in an increase in intracellular cAMP which, in PBMC, induces the suppression of TNF-alpha mRNA transcription and thus cytokine synthesis. Such an effect is antagonistic to that of imiquimod. Furthermore, some TNF-alpha-induced activity, such as cell apoptosis which is dependent on the intracellular cAMP levels might also be affected. Therefore, by counteracting the properties of TNF-alpha and/or its production, the imidazo[1,2-alpha]quinoxalines could be considered as potential anti-inflammatory drugs. The present study was performed to confirm or refute this hypothesis. For this, we characterized the effects of imidazo[1,2-alpha]quinoxalines both on TNF-alpha activity and synthesis in regard to their ability to act as inhibitors of PDE4 (IPDE4). We found that the imidazo[1,2-alpha]quinoxalines dose-dependently prevented the TNF-alpha-triggered death of L929 cells, with the 8-series (-NHCH3 in R4) being the most potent. Moreover, when the effect of the 8-series on TNF-alpha production was investigated using gamma9delta2 T cells, it was observed that these compounds impaired the TCR:CD3-triggered TNF-alpha production. Structure-activity analysis revealed that these properties of the drugs did not coincide with their IPDE4 properties. This prompted further exploration into other signalling mechanisms possibly involved in TNF-alpha action and production, notably the p38 MAPK and the PI3K pathway. We demonstrate here that the imidazo[1,2-alpha]quinoxalines targeted these pathways in a different way: they activated the p38 MAPK pathway whilst inhibiting the PI3K pathway. Such effects on cell signalling could account for the imidazo[1,2-alpha]quinoxalines effects on 1) action and 2) production of TNF-alpha, which define these drugs as potential anti-inflammatory agents.

Extracellular ATP increases cytosolic free calcium in thymocytes and initiates the blastogenesis of the phorbol 12-myristate 13-acetate-treated medullary population.

Exogeneous nucleotides or nucleosides may influence lymphocyte functions such as proliferation and cytotoxicity. We report that ATP, and to a lesser extent ADP, at concentrations as low as 0.3 mM, are highly mitogenic for medullary mature thymocytes, when added in combination with phorbol myristate acetate (PMA), which is only weakly mitogenic by itself. Under the same conditions, the other nucleotides (AMP; GTP, ITP, 2'd-deoxyATP), the non-hydrolysable ATP analogs (p[NH]ppA, pp[CH2]pA) and adenosine are unable to trigger thymocyte blastogenesis. p[NH]ppA, a potent inhibitor of ATP hydrolysis, potentiates the ATP mitogenic effect. In contrast, T-cell-enriched splenocytes do not proliferate in response to ATP + PMA. These data and measurements of interleukin 2 synthesis suggest that ATP may efficiently deliver in thymocytes the calcium signal necessary for the initiation of blastogenesis (in medullary cells). Indeed, among all nucleotides tested, only ATP or ADP were able to increase the intracellular free calcium level in thymocytes, but not in splenocytes. Our results led us to suggest that thymocytes express on their surface receptors specific for ATP, which might be P2 type nucleotide receptors and could be involved in the lymphocyte response through the regulation of intracellular free calcium levels.

Studies of (Na+ + K+)-sensitive ATPase activity in pig lymphocytes. Effects of concanavalin A.

(Na+ + K+)-ATPase activity is demonstrated in plasma membranes from pig mesenteric lymph nodes. After dodecyl sulfate treatment plasma membranes have an 18-fold higher (Na+ + K+)-ATPase activity, while their ouabain-insensitive Mg2+-ATPase is markedly lowered. A solubilized (Na+ +K+)-ATPase fraction, obtained by Lubrol WX treatment of the membranes, has very high specific activity (21 mumol Pi/h per mg protein). Concanavalin A has no effect on these partially purified (Na+ + K+)-ATPase, while inhibits (40%) this activity in less purified fractions which still contain Mg2+-ATPase activity.

Alamethicin or detergent permeabilization of the cell membrane as a tool for adenylate cyclase determination.

Treatment of mouse lymphocytes with very low concentrations of alamethicin or Lubrol PX induces spontaneous permeabilization of the plasma membrane to ATP and allows determination of adenylate cyclase activity in whole cells. The permeabilized cells retain responsiveness to hormones (isoproterenol, adenosine analogs) and to fluoride. The main advantage of this new method is that it does not require any homogenization step, and thus adenylate cyclase activities can be accurately and reproducibly measured with very low amounts of cells. It should be especially useful for the study of purified lymphocyte subpopulations.

Adenosine-induced cyclic AMP increase in pig lymphocytes is not related to adenylate cyclase stimulation.

Adenosine-cyclic AMP relationships have been studied in pig mesenteric lymph node lymphocytes. The early 2--3-fold increase in cyclic AMP accumulation elicited by adenosine and 2-chloroadenosine, an adenosine deaminase-resistant analogue, could not be correlated to similar effects on the adenylate cyclase activity of disrupted cell preparations, but rather to the competitive inhibition of the low Km (0.17 muM) cyclic AMP phosphodiesterase. The existence of adenosine receptors coupled to lymphocyte adenylate cyclase, which had been proposed by several authors, could not be confirmed by this study Adenosine-cyclic AMP relationships do not appear to be involved in concanavalin A stimulation of pig lymphocytes.

Unmasking of membrane enzyme activities and the problem of subcellular localization of adenylate cyclase in pig lymph node lymphocytes.

A large-scale purification of plasma membranes from pig lymph node lymphocytes is described. Centrifugation on a discontinuous sucrose density gradient was performed in a zonal rotor. Adenylate cyclase activity of untreated fractions displayed a profile different from that of plasma membrane enzymatic markers and was maximal at higher density. However, when latent adenylate cyclase was unmasked by Lubrol PX treatment, its maximum was shifted to lower density and was no longer significantly different from that of plasma membrane markers. These results are discussed in terms of cell surface topography.

The proposed 5'-nucleotidase inhibitor in human leukemic cells is an artefact.

It is now well established that human lymphoblastoid cell lines showing immaturity characters display ecto-5'-nucleotidase activities lower than normal levels. A recent paper (Sun, A.S., Holland, J.F. and Ohnuma, T. (1983) Biochim. Biophys. Acta 762, 577-584) mentioned that this phenomenon resulted from the presence of a 5'-nucleotidase inhibitor in these cell lines. We demonstrate here that the use of 5'-[3H]AMP as a substrate, and inadequate analysis of the products formed, led them to a misinterpretation. [3H]Adenosine derived from 5'-[3H]AMP hydrolysis was further transformed into [3H]inosine by the adenosine deaminase activity of the leukemic cell lines tested; [3H]inosine was precipitated with the excess substrate and was not taken into account in the ecto-5'-nucleotidase determination, which led the authors to confuse this adenosine deaminase activity with a 5'-nucleotidase inhibitor. We did not observe 5'-nucleotidase inhibition by leukemic cell cytosol when convenient assay methods were used and showed that the presence of such an inhibitor remains to be established.

Involvement of CD4 in interleukin-6 secretion by U937 monocytic cells stimulated with the lectin jacalin.

The lectin jacalin is mitogenic for CD4 expressing T lymphocytes, interacts with the CD4 molecule, and inhibits HIV infection of CD4+ cells. In the present study the effect of jacalin was tested on cells from the monocyte/macrophage lineage that also express the CD4 molecule. We used CD4+ promyelomonocytic U937 cells differentiated towards the monocytic/macrophage lineage with either a mixture of two physiological agents, retinoic acid (RA) and 1 alpha,25-dihydroxyvitamin D3 (VD), or the exogenous drug phorbol myristate acetate (PMA). The cells resulting from these treatments differed in term of CD4 expression. We focused our attention on interleukin-6 (IL-6) production, which implies an activation of the cells differentiated along both pathways. In CD4+ RA/VD-treated cells, jacalin induced a 10-fold higher IL-6 secretion than did lipopolysaccharide (LPS). This jacalin-induced IL-6 production was inhibited by agents interacting with CD4 (anti-CD4 mAbs and HIV recombinant gp120) or by recombinant soluble CD4. In contrast, the CD4- PMA-differentiated U937 cells did not secrete any IL-6 upon jacalin treatment, while they demonstrated a response to LPS similar to that of the RA/VD-differentiated cells. Together with the fact that jacalin interacts with CD4, these results provide evidence of the involvement of a CD4 dependent pathway in IL-6 production.

The lectin jacalin triggers CD4-mediated lymphocyte signaling by binding CD4 through a protein-protein interaction.

The lectin jacalin specifically stimulates lymphocytes expressing the CD4 antigen. Recent studies have also demonstrated that this lectin interacts with CD4, inhibits in vitro HIV infection, and triggers cell signaling directly via CD4. Jacalin as a lectin was suggested to trigger CD4-mediated cell signaling by interacting with the oligosaccharide side chains of CD4 located on Asn271 and Asn3OO. Such a hypothesis was of importance because it implied that the glycosylated chains could represent a functional domain directly involved in CD4-related cell activation. We analyzed this possibility by studying the effect of hapten sugars on jacalin-induced CD4 cell signaling and jacalin/CD4 interaction, and by studying the binding capacities of the lectin toward glycosylated, deglycosylated, and unglycosylated CD4. The results presented in this study provide evidence that jacalin does not recognize the CD4 oligosaccharide chains and actually binds CD4 through a specific protein-protein interaction; as a consequence these results rule out the involvement of the CD4 saccharide moieties in CD4-mediated cell signaling triggered by the lectin.

Jacalin, a lectin that inhibits in vitro HIV-1 infection, induces intracellular calcium increase via CD4 in cells lacking the CD3/TcR complex.

The lectin jacalin interacts with the CD4 cell surface antigen; this lectin inhibits in vitro infection by human immunodeficiency virus type 1 without preventing virus binding on the host cell. The infection process is known to involve cellular events triggered by the binding of the viral external glycoprotein gp120 to CD4. Herein we demonstrate that jacalin induces cell signaling directly through the CD4 antigen and that independently of the CD3/TcR complex. The capacity of jacalin to trigger cell signals through the CD4 molecule is discussed in relation to its ability to inhibit HIV infection.

A beneficial aspect of a CB1 cannabinoid receptor antagonist: SR141716A is a potent inhibitor of macrophage infection by the intracellular pathogen Brucella suis.

The psychoactive component of marijuana, delta9-tetrahydrocannabinol (THC) suppresses different functions of immunocytes, including the antimicrobicidal activity of macrophages. The triggering of cannabinoid receptors of CB1 and CB2 subtypes present on leukocytes may account for these effects. We investigated the influence of specific CB1 or CB2 receptor antagonists (SR141716A and SR144528, respectively) and nonselective CB1/CB2 cannabinoid receptor agonists (CP55,940 or WIN 55212-2) on macrophage infection by Brucella suis, an intracellular gram-negative bacteria. None of the compounds tested affected bacterial phagocytosis. By contrast, the intracellular multiplication of Brucella was dose-dependently inhibited in cells treated with 10-500 nM SR141716A and 1 microM SR141716A-induced cells exerted a potent microbicidal effect against the bacteria. SR144528, CP55,940, or WIN 55212-2 did not affect (or slightly potentiated) the growth of phagocytized bacteria. However, CP55,940 or WIN 55212-2 reversed the SR141716A-mediated effect, which strongly suggested an involvement of macrophage CB1 receptors in the phenomenon. SR141716A was able to pre-activate macrophages and to trigger an activation signal that inhibited Brucella development. The participation of endogenous cannabinoid ligand(s) in Brucella infection was discussed. Finally, our data show that SR141716A up-regulates the antimicrobial properties of macrophages in vitro and might be a pharmaceutical compound useful for counteracting the development of intramacrophagic gram-negative bacteria.

The [Ca2+]i increase induced in murine thymocytes by extracellular ATP does not involve ATP hydrolysis and is not related to phosphoinositide metabolism.

We have previously demonstrated that exogenous ATP can give medullary thymocytes the calcium message required for the induction of their blastogenesis. In the present study, using the highly sensitive calcium indicator Indo-1, we have measured the effect of exogenous nucleotides on the cytosolic-free calcium concentration [Ca2+]i of thymocytes, and determined inositol phosphate (IP) formation in the same cells, in parallel assays. The results were compared to those obtained with the mitogenic lectin concanavalin A (ConA) in similar experiments. They show that ATP does not mobilize calcium from its internal stores but stimulates its influx from the extracellular medium. Nevertheless, these data do not rule out the possibility that the nucleotide acts through specific P2 purinergic sites.

[5'-Nucleotidase activity of lymphocyte plasma membranes. Effect of concanavalin A]. / Activité 5'-nucléotidase des membranes plasmiques de lymphocytes. Effet de la concanavaline A.

The 5'-nucleotidase properties of isolated lymphocyte plasma membranes from young pig mesenteric nodes are described; nucleosides-5'-monophosphates are the substrates of this specific enzyme. Concanavalin A inhibits this enzyme; on the same membranes this mitogen does not affect alkaline phosphatase and activates the membrane bound (Ca2+) ATPase. The 5'-nucleotidase inhibition is due to a specific interaction of Con A with carbohydrate groups of the membrane; its high positive cooperativity suggests that the lectin promotes reorganization of the membrane bound 5'-nucleotidase. Solubilization of the 5'-nucleotidase does not prevent the effect of Con A and the solubilized enzyme is firmly bound by Con A-Sepharose 4B; these results suggest that Con A inhibits the enzyme by a direct interaction and that 5'-nucleotidase can be considered as an eventual receptor for the lectin.

[Kinetics of Ca 2+ or Mg 2+ activated ATPase from lymphocyte plasma membranes]. / Etude cinétique de l'activité ATPase activée par Ca2+ ou Mg2+ des membranes plasmiques lymphocytaires

The kinetic study of the C2+ ATPase activity of lymphocyte plasma memebranes allowed some properties of this enzyme to be evidenced. The Ca2+-activated hydrolysis of ATP is independent of a non-specific alkaline phosphatase. The substrate of the ATPase activity is the chelate Ca2+- ATP. Mg2+ may substitute for Ca2+ both as chelating ion and as activating ion. Several results suggest that we have only one ATPase, activated either by Ca2+-, or by Mg2+ with less efficiency; both chelates hve the same Km; pH values for maximum activity and transition temperatures are identical; the effects of free ions are also the same, activation at low concentration and inhibition at high concentration.

[Fluorescence study of lymphocyte plasma membranes]. / Etude par fluorescence des membranes plasmiques de lymphocytes

ANS binding parameters--dissociation constant, number of binding sites, rotation freedom--are measured by fluorescence studies of a complex between ANS and lymph node cell plasma membranes. Divalent ions, Mg++ and Ca++, enhance the complex fluorescence intensity without shifting its maximum wavelength : this enhancement is induced by affinity and quantum yield increases, while the number of binding sites remains constant. The complex fluorescence quenching by ethacrynic acid shows the presence of free SH groups in the ANS binding site. An energy transfer takes place between membrane protein tryptophan residues and bound ANS ; the energy transfer yield is unaffected by Ca++ ions. A correlation of these results is postulated with the biological activity of the membrane.

Cell affinity chromatography with ligands immobilized through cleavable mercury-sulfur bonds.

A new methodology for cell separation by affinity chromatography is described. We have conjugated the organomercurial mersalyl to trisacryl beads bearing primary amino groups. Thiolated ligands can be immobilized on this matrix through cleavable Hg-S bonds. Two model studies of cell separation are reported: (i) concanavalin A thiolated with N-succinimidyl-3-(2-pyridyldithio)-propionate and immobilized on mersalyl-trisacryl; mouse thymocytes bound to Con A-mersalyl-trisacryl were eluted from the support by short thiol treatment which preserved cell viability; (ii) anti-dinitrophenyl antibodies modified with S-acetyl-mercaptosuccinic anhydride and immobilized on mersalyl-trisacryl; sheep erythrocytes, previously labelled with trinitrobenzene sulfonic acid, bound to this support and were easily recovered by thiol treatment without hemolysis. This methodology should overcome difficulties frequently encountered in cell affinity chromatography. Cell support multivalent interactions or high affinity of cell-ligand bindings often require drastic elution conditions which prevent viable cell recovery.

Lipoxygenase inhibitors suppress IL-2 synthesis: relationship with rise of [Ca++]i and the events dependent on protein kinase C activation.

The present study was performed in an attempt to understand the mechanism involved in the inhibition of interleukin 2 (IL-2) synthesis by lipoxygenase (LO) pathway inhibitors. Using the two IL-2-producing lymphoid cell lines, (Jurkat and EL4 cells), we showed first that the inhibitory effect of the phenolic compounds tested (NDGA, BHA and caffeic acid) acted on lymphoid cells themselves and not on eventual monocytic or granulocytic contaminant cells. Secondly, these inhibitors were demonstrated as exerting their effect on two levels: they affected the events controlled by both second messengers implicated in T cell activation, namely rise of intracellular free calcium concentration [( Ca++]i) and protein kinase C (PKC) activation. For this purpose, LO inhibitor effects have been compared: (a) on IL-2 production by the two different lines: Jurkat cells, which need both signals, and EL4 cells, which require only PKC activation for the induction of this production; and (b) on the events induced by the different ways of Jurkat cell activation: PHA (or anti-CD3 monoclonal antibody) versus calcium ionophore. These results are discussed with respect to an eventual involvement of arachidonic acid [AA] derivatives in IL-2 synthesis.

PCP and analogs prevent the proliferative response of T lymphocytes by lowering IL2 production. An effect related to the blockade of mitogen-triggered enhancement of free cytosolic calcium concentration.

The psychotomimetic drug PCP displays a vast array of known pharmacological effects, among them its capacity to affect cation transport in nervous and myocardiac tissues. Since increased movements of cations are essential for the immune responses, it has been mentioned that PCP could also depress immune functions by this mechanism. In order to check this hypothesis, we have investigated the effects of PCP and of many other structural derivatives on the blastogenic response of murine or human T lymphocytes. We find that all the drugs block an early event of T lymphocyte activation and prevent their further proliferation; conversely they do not affect primed lymphocytes. The compounds, which do not inhibit interleukin-1 (IL-1) production in stimulated macrophages, lower interleukin-2 (IL-2) synthesis in activated T helper cells. This negative action appears to be related to the inhibition of the rise of free cytosolic calcium concentration [Ca2+]i observed soon after the T receptor triggering and which is an essential message for IL-2 production. The lymphocyte membrane depolarization induced by the drugs could explain the blockade of the lectin-induced [Ca2+]i changes. The study of the structure-activity relationship shows that the PCP analogs which possess a quasi-rigid conformational structure express an inhibitory capacity of T lymphocyte proliferation higher than that of PCP (200 times for some products). Since these compounds interact poorly with the CNS tissues and have few behavioral effects, we suggest that PCP exerts its negative action on lymphocytes on cell components different from its receptor(s) in the CNS.

Amiloride-induced suppression of lymphocyte proliferation: inhibition of IL 2 receptor expression after blockade of early sodium influx.

Increased rates of monovalent cation fluxes are implicated in the activation of lymphocytes by mitogens. Our report shows that amiloride and dimethylamiloride, two inhibitors of the Na+/H+ antiport, dose dependently prevent the proliferation of mitogen-activated human peripheral blood lymphocytes. The action of these drugs follows several mechanisms, since their inhibitory effect can be reversed by extensive washing when they are used at low concentrations (150 microM for amiloride, 40 microM for dimethylamiloride), while at higher non cytotoxic concentrations this reversibility is no longer observed. We have studied the mechanism whereby amiloride inhibits the blastogenesis by measuring their effect on: 1) IL 2 production, 2) acquisition of IL 2 responsiveness and induction of IL 2 receptors, 3) IL 2-induced proliferation. Unlike the expression of IL 2 receptors, IL 2 production and IL 2-dependent proliferation were not inhibited by the low drug concentrations which indeed prevent blastogenesis. Moreover under these conditions, an enhanced accumulation of IL 2 was observed in the supernatants of stimulated cells. These results show that the drugs interact differently with the different cell populations involved in T cell proliferation: increase of an amiloride-dependent sodium influx is an obligatory step required to induce the early increase of the ouabain-dependent potassium influx which is needed for the expression of IL 2 receptors. On the contrary, the influx of potassium necessary for the IL 2-dependent proliferation does not seem to be controlled by the amiloride-dependent sodium flux.

Mechanism whereby ouabain inhibits human T lymphocyte activation: effect on the interleukin 2 pathway.

Through the blockade of the Na-K-ATPase, ouabain inhibits several biochemical and biological events leading to the proliferation of activated lymphocytes. Since we already found that interleukin 1 production was not prevented by ouabain, we investigated by which mechanism this drug inhibits mitogen-induced human T lymphocyte activation, with respect to the interleukin 2 (IL 2) pathway. Our data revealed that at concentrations lower than 0.2 microM, IL 2 accumulation was not reduced in ouabain-treated cultures, even when cell proliferation was completely inhibited (0.1-0.2 microM ouabain). Moreover, in this concentration range, ouabain stimulated in a dose-dependent manner the accumulation of IL 2 in the supernatant of Con A-stimulated lymphocytes (optimum for 0.05 microM corresponding to half inhibition of lymphocyte proliferation). Such an effect, which appears correlated to the inhibition of Na-K-ATPase, suggests a failure of the cell to utilize IL 2. At concentrations higher than 0.3 microM, ouabain inhibited both lymphocyte proliferation and IL 2 production. These observations show that the glycosteroid interacts differently with the different cell populations involved in the cascade of reactions leading to cell proliferation, and suggest that the mitogenic inhibition resulting from the blockade of Na-K-ATPase is not related to the blockade of IL 2 production. On the other hand, we observed that: ouabain inhibited the expression of the receptors for IL 2, an obligatory step in lymphocyte proliferation; ouabain blocked the proliferation of an IL 2 sensitive human T cell line; in both cases the inhibition paralleled that of lymphocyte proliferation. Our data suggest that the essential steps of lymphocyte proliferation in which Na-K-ATPase-dependent K+ fluxes play a critical role are the expression of IL 2 receptors and the IL 2-dependent proliferative step.