Signal Transduction during Platelet-Activating Factor-Induced Lymphocyte Binding to Endothelial Cells

Platelet-activating factor (PAF) is a lipid mediator of inflammation. PAF pretreatment of cultured endothelial cells leads to an increase in lymphocyte binding. We have analysed the intracellular signal transduction during this PAF-induced effect. The protein kinase C activator, phorbol 12-myristate, 13-acetate, mimicked PAF in the binding assay. Concomitantly, the protein kinase C (PKC) inhibitor H7 down-regulated the PAF-induced binding to nearly control level. Also dibutyryl-cAMP treatment of endothelial cells increased lymphocyte binding, but the protein kinase A inhibitor HA1004 did not alter the PAF-induced binding. Furthermore, PAF did not increase the level of cytosolic cAMP in the endothelial cells. Other second messengers, cGMP and Ca2+, had no effect on lymphocyte binding. These findings suggest that protein kinase C, but not other signal transduction pathway, is essential in the PAF-induced lymphocyte binding to endothelial cells.     

Signal transduction during in vitro lymphocyte homing

Lymphocyte binding to endothelium is a necessary prerequisite for lymphocyte homing through endothelium. This is mediated by the binding of ligands on endothelial cells to lymphocyte surface homing receptors. We show in this paper that the intracellular second messenger pathways involved in interferon gamma-induced intercellular adhesion molecule 1 upregulation on endothelial cells are protein kinase C and calcium dependent. Lymphocyte binding to endothelial cells is enhanced by both platelet activating factor and interleukin 1 alpha. Platelet activating factor added to endothelial cultures increases lymphocyte binding within 10 min and operates via protein kinase C but not via cAMP. On the other hand interleukin 1 alpha increases binding within 4 hr and operates via cAMP but not via protein kinase C. These results imply that different mediators of inflammation can activate different signal transduction pathways but lead to similar increases in lymphocyte binding.     

Cocaine-induced increase in the permeability function of human vascular endothelial cell monolayers.

The effects of cocaine on endothelial cell macromolecular transport, electrical resistance, and morphology were assessed. In confluent endothelial monolayers grown on microporus filters, cocaine (0.01 to 1 mmol/L) induced a rapid concentration-dependent increase in permeability to peroxidase and low density lipoprotein. Along with increased transport, the cocaine effect was paralleled by a decrease in transendothelial electrical resistance. Alterations in membrane resistance were fully reversible following washout of the drug, providing evidence that cocaine does not cause permanent injury to the integrity of the monolayer. Cocaines major metabolites, benzoylecgonine and ecgonine methyl ester, had minimal effect on electrical resistance properties, whereas monolayer impedance was markedly depressed by the novel cocaine/alcohol metabolite, cocaine ethyl ester (cocaethylene). Morphologic studies of cocaine-treated endothelial cells revealed a marked disruption of F-actin and the formation of intercellular gaps; no evidence of cell lysis and/or detachment was noted. Forskolin, a potent activator of adenylate cyclase known to promote the endothelial cell barrier function, impaired cocaine-induced changes in electrical resistance and morphology. Cocaine, however, had no effect on resting levels of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) in confluent endothelial monolayers. In summary, the results indicate that cocaine directly induces structural defects in the endothelial cell barrier which enhance the transport of macromolecular tracers, the mechanism does not appear to involve intracellular cAMP.     

The adenylate cyclase toxins

Cyclic AMP is a ubiquitous messenger that integrates many processes of the cell. Diverse families of adenylate cyclases and phosphodiesterases stringently regulate the intracellular concentration of cAMP. Any alteration in the cytosolic concentration of cAMP has a profound effect on the various processes of the cell. Disruption of these cellular processes in vivo is often the most critical event in the pathogenesis of infectious diseases for animals and humans. Many pathogenic bacteria secrete toxins to alter the intracellular concentration of cAMP. These toxins either disrupt the normal regulation of the host cell's adenylate cyclases/phosphodiesterases or they themselves catalyze the synthesis of cAMP in the host cell. The latter are known as the adenylate cyclase toxins. Four such toxins have been identified: the invasive adenylate cyclase of Bordetella pertussis, the edema factor of Bacillus anthracis, ExoY of Pseudomonas aeruginosa, and the adenylate cyclase of Yersinia pestis. These adenylate cyclase toxins enter the eukaryotic host cells and get activated by eukaryotic cofactors, like calmodulin, to trigger the synthesis of cAMP in these cells. By accumulating cAMP in the target cells, these toxins either modulate the cellular function or completely deactivate the cell for further function. The immune effector cells appear to be the primary target of these adenylate cyclase toxins. By accumulating cAMP in the immune effector cells, these adenylate cyclase toxins poison the immune system and thus facilitate the survival of the bacteria in the host.     

A simple solid phase radioimmunoassay specific for human C3b to detect C3b receptors on human lymphoblastoid cell surfaces

Addition of IgG-sensitized human erythrocytes to peripheral blood monocytes elicit a transient increment in monocyte cAMP levels. This increase in cAMP was facilitated when monocytes were preincubated with the phosphodiesterase inhibitors, isobutylmethylxanthine (IBMX) and theophylline, and the adenylate cyclase agonists, isoproterenol and prostaglandin E1 (PGE1). Although these cAMP elevating agents were able to inhibit monocyte ADCC, the degree of inhibition could not be anticipated from the cAMP levels achieved by these drugs since theophylline inhibited monocyte ADCC in doses not elevating cAMP and PGE1, isoproterenol and IBMX were less effective inhibitors of monocyte ADCC than theophylline when comparing their effects on cAMP levels. Both PGE1-induced elevation of cAMP levels and the further increments of cAMP after addition of IgG-sensitized erythrocytes to PGE1-treated monocytes were significantly correlated to the inhibition of beta-glucuronidase release during ADCC. Theophylline in doses of 0.5 mM did not elevate basal levels of monocyte cAMP but facilitated the ADCC-induced cAMP increment concomitant with inhibition of monocyte ADCC and degranulation. Possibly, facilitation of cAMP increments during ADCC by an inhibitory feedback mechanism may be responsible for the inhibition caused by cAMP-elevating agents.     

Dissection of lymphocyte function-associated antigen 1-dependent adhesion and signal transduction in human natural killer cells shown by the use of cholera or pertussis toxin

The effect of the guanosine triphosphate-binding protein (G-protein) inhibitors cholera toxin (Ctx) and pertussis toxin (Ptx) has been analyzed on lymphocyte function-associated antigen 1 (LFA-1)-dependent adhesion and signal transduction in human natural killer (NK) cells. Ctx, but not Ptx, inhibited the LFA-1-dependent adhesion of NK cells to tumor target cells which constitutively express the intercellular cell adhesion molecule-1 (ICAM-1) and to NIH/3T3 mouse fibroblasts stably transfected with human ICAM-1. This effect was detectable only by the use of the entire Ctx but not of the Ctx B subunit. In addition, Ctx could inhibit both NK cell binding and spreading to purified ICAM-1 protein. NK cell treatment with Ctx modified neither the surface expression of LFA-1 nor its Mg²⁺ binding site. These findings, together with the absence of any detectable effect of Ctx on the constitutive phosphorylation of LFA-1α, suggests that this toxin modifies the avidity of LFA-1 for ICAM-1 by acting on LFA-1-cytoskeletal protein association. Unlike Ctx, Ptx did not affect NK cell adhesion. The effects of Ctx and Ptx are unlikely to depend on intracellular levels of cyclic adenosine 3′,5′-monophosphate (cAMP), since a strong increase of cAMP was induced by both toxins. Moreover, this was confirmed by the observation that the LFA-1-dependent adhesion was not inhibited by the adenylate cyclase activator forskolin (FSK), the phosphodiesterase inhibitor isobutyl-1-methylxantine (IBMX), or both, which increase intracellular cAMP levels. Unlike the differential effect on cell adhesion, both the intracellular calcium [Ca²⁺]_i increase and phosphoinositide breakdown mediated via LFA-1 were consistently inhibited in a dose-dependent manner by both Ctx and Ptx. Also in this case, the inhibitory effect did not depend on an increase of intracellular cAMP as indicated by NK cell treatment with FSK, IBMX, or both. Further evidence of the involvement of G-proteins in LFA-1-mediated signal transduction was the inhibitory effect of the GDP analog guanosine-5′-O-2-thiodiphosphate (GDPßS) on LFA-1-mediated calcium mobilization. Taken together, our data provide evidence that the LFA-1-mediated NK cell adhesion and signal transduction are partially independent phenomena which may be regulated by different G-proteins.     

Involvement of adenylate cyclase and p70S6-kinase activation in IL-10 up-regulation in human monocytes by gp41 envelope protein of human immunodeficiency virus type 1

Our previous results show that recombinant gp41 (aa565–647), the extracellular domain of HIV-1 transmembrane glycoprotein, stimulates interleukin-10 (IL-10) production in human monocytes. The signal cascade transducing this effect is not yet clear. In this study, we examined whether gp41-induced IL-10 up-regulation is mediated by the previously described synergistic activation of cAMP and NF-κB pathways. gp41 induced cAMP accumulation in monocytes in a time- and concentration-dependent manner and the adenylate cyclase inhibitor SQ 22536 suppressed gp41-induced IL-10 production in monocytes. In contrast, gp41 failed to stimulate NF-κB binding activity in as much as no NF-κB bound to the main NF-κB-binding site 2 of the IL-10 promoter after addition of gp41. We also examined the involvement of other signal transduction pathways. Specific inhibitors of p70^S6-kinase (rapamycin), and G_i protein (pertussis toxin), prevented induction of IL-10 production by gp41 in monocytes, while inhibitors of the phosphatidylinositol 3-kinase (PI 3-kinase) (wortmannin) and mitogen-activated protein kinase (MAPK) pathway (PD 98059) did not. Thus HIV-1 gp41-induced IL-10 up-regulation in monocytes may not involve NF-κB, MAPK, or PI 3-kinase activation, but rather may operate through activation of adenylate cyclase and pertussis-toxin-sensitive G_i/G_o protein to effect p70^S6-kinase activation. Received: 28 September 1998 / Received after revision: 27 October 1998 / Accepted: 3 November 1998     

Inhibition of Lymphocyte-Mediated Cytolysis and Cyclic amp Phosphodiesterase by Erythro-9–(2-Hydroxy-3-Nonyl)Adenine

Abstract

The adenosine deaminase (ADA) inhibitor erythro-9–(2-hydroxy-3-nonyl)adenine (EHNA), at low concentrations (<10 µM), enhances the inhibitory activity of adenosine against lymphocyte-mediated cytolysis (LMC) without itself being inhibitory. At higher concentrations, EHNA alone is inhibitory to LMC with an IC₅₀ of 160 µM. This inhibition is reversible upon washout, appears to affect an early stage of the lytic process, and does not appear to involve changes in basal levels of cyclic AMP (cAMP), ribonucleoside 5′-triphosphate pool sizes, S-adenosylhomocysteine levels, or protein carboxy-methylation. EHNA does enhance the cAMP response of cytolytic lymphocytes (CL) to activators of adenylate cyclase such as prostaglandin E_l. EHNA inhibits lymphocyte high-affinity cAMP phosphodiesterase at immunosuppressive levels, exhibiting hyperbolic mixed-type inhibition (K_i = 83 µM, α = 0.47, β = 0.18). Whereas inhibition of intralymphocytic ADA is complete at low concentrations (<25 µM) of EHNA, inhibition of LMC and intralymphocytic cAMP phosphodiesterase increases linearly with EHNA concentration to at least 200 µM. The presence of 200 µM EHNA during the centrifugation of mixtures of CL and EL4 leukemia target cells leads to increased CL cAMP levels. 2′-Deoxycoformycin, a more potent ADA inhibitor than EHNA, is not inhibitory to LMC and shows none of these cAMP-related effects. These results suggest that CL-target cell contact stimulates adenylate cyclase in the CL and that EHNA inhibits LMC due to its enhancement of this target cell-stimulated elevation of cAMP.     

Characterization of the role for calcium influx in mitogen-induced triggering of human T cells. Identification of calcium-dependent and calcium-independent signals

Entry of Ca2+ into the cell is recognized as an important activation signal for mitogen-induced lymphocyte proliferation. Changes in free cytosolic Ca2+ [( Ca2+]i) can now be measured directly using the fluorescent reagent quin-2. To analyze the role of [Ca2+]i in human T cell activation, we have determined the effect of the calcium channel blocker, nifedipine, on phytohemagglutinin (PHA)-induced lymphocyte proliferation. At a concentration of 50 microM, nifedipine is nontoxic, and prevents PHA-induced proliferation. In parallel the drug prevents the lectin-induced increase in concentration of [Ca2+]i and interleukin 2 (IL2) secretion; IL2 receptor expression is unaffected. In the presence of exogenous IL2, cell proliferation proceeds normally. Treatment of the cells with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate prevents the inhibitory effect of nifedipine on cell proliferation. Since TPA is itself nonmitogenic and does not affect levels of cytosolic Ca2+, these data and the data on IL2 receptor expression indicate that PHA can generate an activation signal(s) which is [Ca2+]i independent.     

Lymphocyte Binding to and Penetration through Vascular Endothelium is Stimulated by Platelet-Activating Factor

Lymphocytes have an important role in acute inflammatory reactions such as acute allograft rejection. Recirculating lymphocytes attach to vascular endothelium and penetrate through it into tissue parenchyma. Many recent studies have shown that different protein mediators, like gamma interferon, interleukin 1, and tumour necrosis factor enhance lymphocyte binding to and penetration through the endothelium, i.e. lymphocyte homing. We investigated the effect of platelet-activating factor (PAF) in lymphocyte binding and penetration in vitro. Treatment of rat heart microvascular endothelial monolayers with PAF (10(-6)-10(-10) M) increased the lymphocyte binding up to 1.6-fold. The effect is dose- and time-dependent. The PAF effect was reversible upon removal of the agonist: 60 min after removal of PAF no increase in the lymphocyte binding was detected. Pretreatment of endothelial cells, lymphocytes, or both of these cell types led to an increase in lymphocyte binding to endothelial monolayers. The effect of PAF in lymphocyte penetration through endothelium was investigated by using endothelial cell monolayers cultured on top of millipore filters. An optimal PAF dose (10(-8) M) for 10 min increased the number of lymphocytes penetrating through the endothelial cell monolayer into the filter by a factor of 3. These results suggest that PAF has no important role in lymphocyte homing, since it can activate the endothelial cells, the lymphocytes, or both cell types.     

Cellular regulation of islet hormone secretion by the incretin hormone glucagon-like peptide 1

 Glucagon-like peptide 1 is a gastrointestinally derived hormone with profound effects on nutrient-induced pancreatic hormone release. GLP-1 modulates insulin, glucagon and somatostatin secretion by binding to guanine nucleotide binding protein-coupled receptors resulting in the activation of adenylate cyclase and generation of cyclic adenosine monophosphate (cAMP). In the B-cell, cAMP, via activation of protein kinase A, interacts with a plethora of signal transduction processes including ion channel activity, intracellular Ca²⁺ handling and exocytosis of the insulin-containing granules. The stimulatory action of GLP-1 on insulin secretion, contrary to that of the currently used hypoglycaemic sulphonylureas, is glucose dependent and requires the presence of normal or elevated concentrations of the sugar. For this reason, GLP-1 attracts much interest as a possible novel principle for the treatment of human type-2 diabetes. Here we review the actions of GLP-1 on islet cell function and attempt to integrate current knowledge into a working model for the control of pancreatic hormone secretion.     

Components of the intracellular cAMP system supporting the olfactory reception of amyl alcohol

Experiments on isolated frog olfactory epithelium, using vital luminescent microscopy showed that the olfactory transduction of amyl alcohol is mediated by the intracellular cAMP signaling system. Increases in intracellular cAMP levels resulted from activation of adenylate cyclase type III via odorant-induced stimulation of G protein linked to it.     

Inhibition of Human NK Cell Cytotoxicity by Induction of Cyclic AMP Depends on Impaired Target Cell Recognition

Induction of cyclic AMP (cAMP) depresses natural killer (NK) cell activity. The present results demonstrate that this is dependent on a decreased capacity of the effector cells to conjugate to target cells. This was found either if dibutyryl-cAMP was used or if cAMP was induced by adenylate cyclase stimulation with prostaglandin E1 (PGE1) or by inhibition of phosphodiesterase activity with the inhibitor ZK 62711. The sites of action for cAMP-induced NK suppression and interferon (IFN)-induced NK enhancement are demonstrated to be distinct, since IFN acts by increasing the lytic efficiency and the recycling capacity without influencing target binding. Sequential treatment with cAMP/IFN and IFN/cAMP shows that IFN can neither restore target binding when added after cAMP nor protect against the cAMP-induced target binding inhibition when added before cAMP. The results are discussed in view of earlier data on cAMP in relation to cell membrane functions and cellular recognition, the mechanism underlying the cAMP-induced target binding inhibition, and the potential of the NK system as an indicator for immunosuppression. The present work also demonstrates the particular subpopulation in peripheral blood which mediates most NK activity, to respond strongly to PGE1 stimulation with regard to cAMP induction.     

FSH and testosterone effects in seminiferous tubules of immature hypophysectomized rats

The effects of follicle-stimulating hormone (FSH) and testosterone on the development of the cytosolic germ cell adenylate cyclase and germ cell morphology in rats hypophysectomized at 29 days of age were studied. Following hypophysectomy, the adenylate cyclase content fell to marginal levels and germ cell development ceased at the late pachytene stage. Testosterone treatment led to a moderate increase in the cytosolic enzyme content and to progression of spermatid cell development to stages 8-12. FSH treatment with doses of 80-100 micrograms/day restored enzyme content to levels seen in control rats, as well as progression of germ cell development up to stages 15-16, i.e., to the same stages present in age-matched control (sham-operated) rats. The results indicate that in immature rats FSH is essential for spermatid cell maturation as is evidenced by its ability to stimulate the formation of cytosolic germ cell adenylate cyclase to quantitatively normal levels, as well as to stimulate the development of spermatid cells.     

The adenylate cyclase toxin contributes to the survival of Bordetella pertussis within human macrophages

The adenylate cyclase toxin (ACT) of Bordetella pertussis has been shown to penetrate eucaryotic cells and produce a rapid elevation in intracellular cAMP which leads to altered cell function. Recent studies have demonstrated an intracellular state for the bacteria within professional and non-professional phagocytes. A virulent strain was compared to two ACT defective strains to determine if this toxin contributes to intracellular survival within human macrophages. When challenged by 10⁶ macrophages/ml in a cell invasion assay, 10³ bacteria/ml were recovered from samples containing the ACT defective strains. These values were two log units less than the number of bacteria recovered from samples containing the isogenic parent. The binding and uptake of all strains by the macrophages were equivalent, suggesting that ACT does not affect adhesion nor endocytosis but rather protects against macrophage killing following uptake. Drug-induced elevation of cAMP levels within the macrophage by forskolin increased the number of surviving bacteria in samples containing the mutant strains to values equal to those obtained with the parent strain. Therefore, the protective effect conveyed by ACT is the result of toxin-induced elevation of cAMP within the macrophage concomitant with bacterial uptake.     

Effects of hypoxia on cyclic nucleotide formation in rabbit carotid body in vitro

The present experiments measured cAMP and cGMP in the arterial chemosensory tissue of the rabbit carotid body exposed for 10 min in vitro to normoxic or hypoxic conditions, or to specific activators of adenylate cyclase (forskolin) and guanylate cyclase (sodium nitroprusside). The enzyme activators elevated the basal levels of cAMP (48 x) and cGMP (3.7 x), respectively. Hypoxic media increased cAMP in the carotid body by 3.6-fold, but the levels of cGMP were reduced by 33% in media equilibrated with low O2. The data are consistent with the notion that cyclic nucleotides are involved in the transduction of natural stimuli and/or the neurotransmitter feedback modulation of chemosensory type I cells.     

Adenylate cyclase, cyclic AMP and IgE-mediated desensitization in rat mast cells

Rat peritoneal mast cells were desensitized up to 100% with 1-4 additions of suboptimal concentrations of anti-rat IgE over 60-90 min. Desensitized cells rechallenged with anti-IgE showed a 2- to 10-fold increase in cAMP during the initial 1-5 min. Membrane preparations from desensitized cells showed up to a 100-fold rise in cyclase activity following incubation with anti-IgE, compared to a 5- to 10-fold rise in control cells. Control and desensitized cell membranes rechallenged with anti-IgE showed nearly identical levels of phosphodiesterase activation. We conclude that following desensitization, repetitive low levels of anti-IgE binding induce atypically high and sustained activation of adenylate cyclase which may reflect primary or secondary regulatory events in the development of desensitization.     

The adrenergic system in lymphocytes from children with cystic fibrosis

Summary Several in vivo and in vitro studies have suggested that children suffering from cystic fibrosis (CF) might have a general defect of beta-adrenoceptors on the cell surface which might account for an unbalanced secretory process. In order to investigate if this view holds true, we determined the beta-adrenoceptor density and affinity on lymphocytes by means of radioligand studies using 125-iodo-cyano-pindolol (125-ICYP) in 20 children with CF. Cyclic AMP (cAMP) response was also investigated after specific beta-adrenoceptor stimulation with isoprenaline (IPN) and after direct stimulation of the adenylate cyclase with forskolin in lymphocytes. Children with CF and controls have identical numbers and affinities of beta-adrenoceptors on lymphocytes. The cyclic AMP response was identical in CF- and in age-matched control children regardless whether adenylate cyclase was stimulated directly or via beta-adrenoceptors. In conclusion, the data support the view that no general adrenoceptor or adenylate cyclase defect exists in CF. As several studies have found abnormal reactions to adrenergic stimuli in CF patients, we presume that there is a defect beyond the level of adrenergic receptors and cAMP which remains to be identified.Abbreviations BS/Ly binding sites per lymphocyte - BSA bovine serum albumin - cAMP cyclic AMP - CF Cystic Fibrosis - 125-ICYP 125-iodo-cyano-pindolol - IPN isoprenaline - SEM standard error of mean