Regulation of Fc gamma receptor expression and phagocytosis of a human monoblast cell line U937. Participation of cAMP and protein kinase C in the effects of IFN-gamma and phorbol ester

We investigated the positive and negative effects of IFN-gamma, PMA, dibutyryl cAMP (Bt2cAMP), dexamethasone and transforming growth factor-beta (TGF-beta) on Fc gamma R subtype expression and phagocytosis of a human monoblast cell line, U937. IFN-gamma increased and Bt2cAMP decreased Fc gamma RI expression determined by a mAb 32.2, whereas PMA and Bt2cAMP increased Fc gamma RII expression determined by a mAb IV-3. Phagocytosis was measured microscopically by counting ingested aggregated human IgG- or BSA-treated ox E (Eo'-IgG or Eo'-BSA). IFN-gamma increased the phagocytosis of Eo'-IgG but not that of Eo'-BSA, and PMA increased the phagocytosis of both Eo'-IgG and Eo'-BSA. Bt2cAMP decreased both basal and IFN-gamma- and PMA-augmented phagocytosis of U937 cells. Dexamethasone also inhibited both basal and IFN-gamma-augmented Fc gamma RI expression and PMA-augmented Fc gamma RII expression and phagocytosis, but did not affect IFN-gamma-augmented phagocytosis of Eo'-IgG. The augmentation of phagocytosis of Eo'-IgG by IFN-gamma thus seems to be due mainly to the increased internalizing process rather than to increased Fc gamma RI expression. TGF-beta slightly decreased Fc gamma R expression. In a study of the participation of protein kinase C (PK-C), it was found that H-7, a PK-C inhibitor, did not inhibit either IFN-gamma- or PMA-enhanced Fc gamma RI and Fc gamma RII expression, respectively, and 1-oleoyl-2-acetylglycerol and N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide, both PK-C activators, did not show any apparent increase in Fc gamma R expression and phagocytosis. These results show that Fc gamma RI and Fc gamma RII expression on U937 cells is regulated by different mechanisms and that IFN-gamma and PMA play their roles in Fc gamma R expression and phagocytosis by different pathways. It is possible that cAMP but not PK-C plays an important role in the regulation of Fc gamma R expression and phagocytosis.     

Effect of insulin and isoproterenol on macrophage phagocytic activity

To reveal the influence of cyclic adenosine monophosphate (cAMP) on the completion of the phagocytosis of salmonellae, the influence of insulin and isoproterenol on the phagocytic activity of peritoneal macrophages obtained from mice infected with S. typhimurium strains differing in virulence was studied in vitro. The study showed that isoproterenol, while increasing the intracellular content of cAMP, suppressed the bactericidal properties of macrophages with respect to salmonellae, whereas insulin decreased the level of cAMP in the cells and thus facilitated more rapid and complete digestion of ingested bacteria irrespective of their virulence.     

Transient increase in cyclic AMP localized to macrophage phagosomes

Cyclic AMP (cAMP) regulates many biological processes and cellular functions. The importance of spatially localized intracellular gradients of cAMP is increasingly appreciated. Previous work in macrophages has shown that cAMP is produced during phagocytosis and that elevated cAMP levels suppress host defense functions, including generation of proinflammatory mediators, phagocytosis and killing. However, the spatial and kinetic characteristics of cAMP generation in phagocytosing macrophages have yet to be examined. Using a Förster resonance energy transfer (FRET)-based cAMP biosensor, we measured the generation of cAMP in live macrophages. We detected no difference in bulk intracellular cAMP levels between resting cells and cells actively phagocytosing IgG-opsonized particles. However, analysis with the biosensor revealed a rapid decrease in FRET signal corresponding to a transient burst of cAMP production localized to the forming phagosome. cAMP levels returned to baseline after the particle was internalized. These studies indicate that localized increases in cAMP accompany phagosome formation and provide a framework for a more complete understanding of how cAMP regulates macrophage host defense functions.     

DNA polymerase alpha, beta and gamma activities in rabbit spleen cell populations stimulated by various doses of concanavalin A

Adherence and phagocytosis of ⁵¹chromium labeled sheep red blood cells ([⁵¹Cr]-sRBC) by P388 D₁ cells in tissue culture were studied under various conditions and were found to possess certain requirements including opsonization, temperature, microfilaments and cyclic nucleotide levels. Exogenous administration of 10^?2 M N⁶, O²-dibutyryl adenosine 3′–5′ cyclic monophosphoric acid (db-cAMP) or adenosine 3′–5′ cyclic monophosphoric acid (cAMP) inhibited phagocytosis of opsonized [⁵¹Cr]-sRBC by 36 and 42%, respectively. Aminophylline potentiated the inhibitory response to both cAMP and db-cAMP. The measurement of endogenous cyclic nucleotide levels during phagocytosis of opsonized sRBC showed a rise in guanosine 3′–5′ cyclic monophosphate (cGMP) during the first 5 min with a gradual decline to control levels at 45 min and a rise in cAMP levels reaching a peak at 30 min which remained above control values for the duration of the experiment. As the rate of phagocytosis decreased the ratio of $\text{cAMP}\text{cGMP}$ increased. These observations emphasize the importance of metabolic functions and cyclic nucleotides during phagocytosis by the P388 D₁ cells and strengthen the usefulness of the P388 D₁ cells as a model in evaluating various macrophage activities.     

Compartmentalization of PDE-4 and cAMP-dependent protein kinase in neutrophils and macrophages during phagocytosis

The compartmentalization of cAMP in human neutrophils during phagocytosis of serum-opsonized zymosan suggests that cAMP is an important second messenger for regulating phagocytosis. Type 4 cAMP-specific phosphodiesterase (PDE-4), cAMP-dependent protein kinase (PKA), and adenylate cyclase are the principal effector molecules for cAMP regulation in phagocytes. Immunofluorescence microscopy demonstrated that PDE-4 isoforms (HSPDE-4A, HSPDE-4B, HSPDE-4D) were targeted to the forming phagosome in neutrophils, and were colocalized with the catalytic subunit of PKA and degranulated myeloperoxidase. Phagocytosis and accumulation of PDE-4 and PKA near adherent zymosan were inhibited by elevating cAMP levels with forskolin or rolipram. cAMP, PDE-4, and PKA were localized at sites of zymosan adherence in cells treated with cytochalasin D to inhibit phagosome formation, suggesting that zymosan engagement to Fc/CR3 receptors triggers cAMP elevations at sites of phagocytosis. HSPDE-4A, HSPDE-4B, HSPDE-4D, and PKA also were localized at the forming phagosome in monocyte-derived macrophages, and the lysosomal marker CD63 demonstrated the absence of PDE-4 around internalized phagolysosomes. These results suggest that cAMP levels are focally regulated by PDE-4 at the nascent phagosome, and that PKA may phosphorylate proteins associated with pseudopodia formation and phagosome internalization.     

Modulation of cyclic AMP in purified rat mast cells. I. Responses to pharmacologic, metabolic, and physical stimuli.

The cyclic adenosine 3', 5'-monophosphate (cAMP) content of isolated unstimulated mast cells and the changes induced by a variety of pharmacologic, metabolic, and physical stimuli were studied. A modified bovine serum albumin density gradient purification method consistently provided mast cell preparations which were 95% or more pure, without apparent damage, and a 73% recovery of the mast cells applied to the gradients. The measured cAMP in unstimulated mast cells was high, a mean of 16 picomoles per million cells. Moderate agitation or contact with glass increased cAMP content about 2-fold. When calcium was omitted from the medium mast cell cAMP was markedly elevated; incremental increases in added calcium ion concentration from 1 muM to 1 mM caused a linear decrease in cAMP content. Theophylline (3 to 20 mM) caused a dose-related increase in mast cell cAMP content, approximately 2-fold at 20 mM theophylline. Epinephrine (0.01 to 1 mM) caused a modest, dose-related increase in cAMP. In the presence of theophylline, epinephrine increased cAMP levels equal to or greater than the sum of the effects of the agents used individually. The increase in cAMP induced by epinephrine was completely inhibited by 100 muM propranolol and partially inhibited by 10 muM propranolol, thus suggesting that a beta adrenergic receptor is involved. Prostaglandin E1 (PGE1) and histamine (in the presence of theophylline) also raised cAMP. Carbamylcholine (1 nM) lowered cAMP 38%; Atropine (0.1 mM) inhibited the decrease in cAMP induced by 1 nM carbamylcholine by 83% indicating that a muscarinic receptor is involved. In these homogeneous single cell suspensions, therefore, cholinergic and beta adrenergic agents have opposing effects on cAMP levels. Diazoxide (10 muM) and adenine (1 muM) caused 37 and 32% decreases in cAMP, respectively. The availability of highly purified mast cells and the identification of agents which either decrease or increase cAMP content allows a direct examination of the role of cAMP in histamine release.     

Cyclic AMP and its functional relationship in Tetrahymena: a comparison between phagocytosis and glucose uptake.

In Tetrahymena, an increase in the level of cAMP is accompanied by an increased phagocytotic rate, whereas increased sugar uptake is parallelled by a decreased cAMP level. The increase in cAMP level seems to be decisive with respect to phagocytosis as a basic phenomenon of life. In the action of epinephrine, however, some mechanism other than cAMP mediation may be involved. Depending on concentration, one hormone may provoke either an increase or a decrease in cAMP level, and this in turn triggers the corresponding function.     

Response of a dog kidney cell line (MDCK) to antidiuretic hormone (ADH) with special reference to activation of protein kinase

Antidiuretic hormone (ADH) increased the adenosine 3′,5′-cyclic monophosphate (cAMP) content of cultured cells of the MDCK line, derived from normal dog kidney, after activation of adenylate cyclase. Parathyroid hormone and insulin did not affect the cAMP content of the cells and ADH did not increase the cAMP content of HeLa or 3T6 cells. Thus the effect of ADH on MDCK cells was specific. ADH activated the protein kinase of MDCK cells. Conversion of cAMP-dependent protein kinase to a cAMP-independent catalytic subunit in the ADH-treated cells was demonstrated by glycerol density gradient centrifugation.     

Influence of aminophylline on the lipids in Microsporum gypseum.

The effect of aminophylline on the lipid synthesis of Microsporum gypseum has been examined. A decreased incorporation of [14C]acetate into lipids was observed when the cells were incubated for 1 h with aminophylline which was reflected in all the individual lipid fractions. However, cells grown with aminophylline in the growth medium exhibited increased levels of total phospholipids, which was probably due to a rise in intracellular cAMP as these cells exhibited 4-fold increased levels of cAMP. Decreased activity of phosphodiesterase by aminophylline accounts for the increased cAMP levels. Increased phospholipid content in aminophylline grown cells was further supported by the increased incorporation of [14C]acetate into phospholipids as well as increased activities of phospholipid biosynthetic enzymes in comparison to non-supplemented cells.     

低氧对CRF,AVP和NE刺激体外培养腺垂体细胞生成cAMP的影响

本文探讨了ＣＲＦ,ＡＶＰ及ＮＥ对体外培养大鼠腺垂体细胞生成ｃＡＭＰ的作用。ＣＲＦ刺激体外培养大鼠腺垂体细胞内ｃＡＭＰ的生成,且浓度与效应正相关。ＡＶＰ未引起细胞内ｃＡＭＰ差异性变化（Ｐ＞０．５）。ＮＥ使培养腺垂体细胞内ｃＡＭＰ水平降低。低氧使ＣＲＦ刺激ｃＡＭＰ生成的作用降低。而ＡＶＰ及ＮＥ可能是通过其它胞内信息通路。     

Alterations of glial tumor cell Ca2+ metabolism and Ca2+-dependent cAMP accumulation by phorbol myristate acetate

C6 glial tumor cells exposed to phorbol myristate acetate (PMA) possessed lowered cAMP content, reduced ability to accumulate cAMP in response to norepinephrine or cholera toxin, and a 3-fold increase in the concentration of norepinephrine producing 50% of the maximal rate of cAMP accumulation. Detectable effects on cAMP accumulation occurred within 10 min of exposure to PMA, and prominent effects by 2 h. PMA similarly affected cells pretreated with cycloheximide. In contrast, Ca2+-depleted preparations of control and PMA-treated cells accumulated cAMP identically in response to norepinephrine or cholera toxin. Ca2+ restoration, which increased the rate of cAMP accumulation in control cells severalfold, did not enhance cAMP accumulation in PMA-treated cells. Neither high catecholamine nor high extracellular Ca2+ concentrations reversed the suppression of cAMP accumulation by PMA. Trifluoperazine, which inhibited the Ca2+-dependent component of norepinephrine-stimulated cAMP accumulation in control cells, did not significantly reduce norepinephrine-stimulated cAMP accumulation in PMA-treated cells. Cell free preparations of control and PMA-treated cultures did not differ significantly in calmodulin content or in Ca2+-stimulated adenylate cyclase, Ca2+-dependent cAMP phosphodiesterase, and (Ca2+-Mg2+)-ATPase activities. The Ca2+ content, however, of intact cells decreased with time of PMA treatment. Within minutes after exposure to PMA, the ability of Ca2+-depleted cells to take up 45Ca was significantly reduced. Both 45Ca uptake and Ca2+-dependent cAMP accumulation were reduced over the same PMA concentration range.     

Direct stimulation of adenylate cyclase by mechanical forces in S49 mouse lymphoma cells during hyposmotic swelling.

S49 mouse lymphoma cells respond to swelling deformation with rapid increases in intracellular calcium and cAMP. Experiments demonstrate that these increases in calcium and cAMP concentrations are not coupled in a regulatory manner. Direct inhibition of adenylate cyclase in wild type cells with miconazole prevented swelling-induced accumulation of cAMP. No effect of swelling was observed on the activity of cAMP phosphodiesterase. Additionally, complete inhibition of cAMP phosphodiesterase did not prevent swelling-induced cAMP accumulation. Experiments involving cyc- mutants (lacking the Gs-alpha protein) and 2',5'-dideoxyadenosine indicate that increased adenylate cyclase activity with swelling is not mediated by Gs. No evidence was found for attenuation of Gi-mediated inhibition of adenylate cyclase activity following swelling. In addition, exposure to pertussis toxin or phorbol ester, which disrupts Gi inhibition of adenylate cyclase did not prevent cAMP accumulation following swelling. Disruption of the actin membrane skeleton resulted in a significant accumulation of cAMP which was not further increased by swelling. Disruption of the microtubular cytoskeleton also increased cAMP content in S49 cells which could be further increased by swelling. It is concluded that S49 cell-adenylate cyclase responds directly to mechanical forces transmitted through the actin membrane skeleton.     

Leptin Enhances Availability of Apoptotic Cell-Derived Self-Antigen in Systemic Lupus Erythematosus

In systemic lupus erythematosus (SLE), the availability of self-antigen promotes and fuels self-reactive immune responses. Apoptotic cells represent a major source of self-antigens, and an impairment of the removal of apoptotic material containing self-antigen can contribute to the development of autoimmunity. To address whether the adipocytokine leptin - which favors autoimmune responses through little understood mechanisms - could modulate the handling of apoptotic cells in SLE, we evaluated the ability of leptin to modulate the capacity of macrophages to phagocytose apoptotic bodies in (NZB×NZW)F₁ lupus mice. It was found that leptin promoted phagocytosis of apoptotic cells by macrophages by modulating cAMP levels in macrophages. This finding associated with an increased availability of antigen that favored the development of T cell responses to apoptotic-derived antigen. As leptin promotes macrophage phagocytosis of apoptotic bodies in SLE and subsequent availability of apoptotic-derived antigen to T cells, an inhibition of this process via leptin blockade might have a therapeutic potential in SLE.     

Blue light stimulates cyanobacterial motility via a cAMP signal transduction system

The participation of cAMP in photosignal transduction in cyanobacteria was investigated. When cells of the cyanobacterium Synechocystis sp. PCC 6803 were exposed to light, cellular cAMP contents increased within a few minutes. Among incident monochromatic lights, blue light (450 nm) markedly increased cellular cAMP content, while red (630 nm) and far-red (720 nm) lights did not. Disruption of the cya1 gene encoding an adenylate cyclase caused the insensitivity of cellular cAMP level to blue light. Treatment of wild-type cells with the flavin antagonist phenylacetic acid inhibited this blue light effect. The motility of wild-type cells was enhanced by blue light, whereas that of cya1 mutant cells was not. Based on these results, we concluded that a blue light-cAMP signal transduction system stimulates the motility of Synechocystis sp. PCC 6803.     

Correlation between intracellular cAMP levels and phospholipids of Microsporum gypseum.

Atropine, a modulator of cAMP has been used to examine the relationship between phospholipids and intracellular levels of cAMP in Microsporum gypseum. A decreased phospholipid content was observed in atropine grown cells as a result of reduced levels of intracellular cAMP. This decline was caused by the inhibitory effect of atropine on adenylate cyclase. Lowered phospholipid content was supported by decreased [14C]acetate incorporation as well as reduced activities of key enzymes of phospholipid biosynthesis. In vitro supplementation of atropine in control cells also caused inhibition in lipid synthesis indicating similar effects of atropine and its metabolites. These results in conjunction with our previous report, in which enhanced levels of cAMP resulted in increased phospholipid synthesis, suggest a direct correlation between phospholipid biosynthesis and intracellular levels of cAMP in M. gypseum.     

The effect of chronic fatty acid treatment on lipolysis in 3T3-L1 adipocytes.

Various saturated and unsaturated fatty acids were included in the culture medium to test their effects on lipolysis in 3T3-L1 adipocytes. Following prolonged incubation, only oleate was found to exert enhancing effect on basal and isoproterenol-stimulated lipolysis. The effect of oleate was concentration-dependent and was accompanied with increased intracellular cAMP content. Furthermore, the lipolytic response induced by isobutyl-methylxanthine, forskolin or dibutyryl cAMP was also increased in adipocytes treated with oleate. Thus, it appears that in addition to an increased cAMP accumulation, a step distal to cAMP production in the cells may be involved in inducing enhanced lipolysis in 3T3-L1 adipocytes by prolonged exposure to oleate.     

Combined activity of post‐exercise concentrations of NA and eHsp72 on human neutrophil function: Role of cAMP

Extracellular heat shock proteins of 72 kDa (eHsp72) and noradrenaline (NA) can act as “danger signals” during exercise‐induced stress by activating neutrophil function (chemotaxis, phagocytosis, and fungicidal capacity). In addition, post‐exercise concentrations of NA increase the expression and release of Hsp72 by human neutrophils, and adrenoreceptors and cAMP are involved in the stimulation of neutrophils by eHsp72. This suggests an interaction between the two molecules in the modulation of neutrophils during exercise‐induced stress. Given this context, the aim of the present investigation was to study the combined activity of post‐exercise circulating concentrations of NA and eHsp72 on the neutrophil phagocytic process, and to evaluate the role of cAMP as intracellular signal in these effects. Results showed an accumulative stimulation of chemotaxis induced by NA and eHsp72. However, while NA and eHsp72, separately, stimulate the phagocytosis and fungicidal activity of neutrophils, when they act together they do not modify these capacities of neutrophils. Similarly, post‐exercise concentrations of NA and eHsp72 separately increased the intracellular level of cAMP, but NA and eHsp72 acting together did not modify the intracellular concentration of cAMP. These results confirm that cAMP can be involved in the autocrine/paracrine physiological regulation of phagocytosis and fungicidal capacity of human neutrophils mediated by NA and eHsp72 in the context of exercise‐induced stress. J. Cell. Physiol. 228: 1902–1906, 2013. © 2013 Wiley Periodicals, Inc.     

Perinatal activity of the hypothalamic-pituitary-gonadal axis in the lamb. II. In vitro testicular response to human chorionic gonadotropin and choleratoxin in the first 2 months of life

Testicular response to human chorionic gonadotropin (hCG) was studied in male lambs. Adenosine 3':5'-cyclic monophosphate (cAMP), testosterone (T), delta 4-androstenedione and 17 alpha-hydroxyprogesterone content and cAMP and T production by dispersed interstitial cells were assessed in control and hCG-pretreated animals. Plasma T levels increased after hCG at 1, 4 and 8 weeks. Increments in the testicular content of cAMP, delta 4-androstenedione, and T were greater at 8 weeks and that of 17 alpha-hydroxyprogesterone and 125I-hCG binding to dispersed interstitial cells were identical at all ages. cAMP and T production by dispersed interstitial cells from nonstimulated animals and the response to hCG and choleratoxin were similar in all lambs. In contrast, cAMP and T production were higher at 1 week only in animals pretreated with hCG in vivo. These data are compatible with hCG-induced desensitization at 4 and 8 weeks.     

Molecular regulation of phagocyte function. Evidence for involvement of a guanosine triphosphate-binding protein in opsonin-mediated phagocytosis by monocytes

Although many functions of phagocytes are known to be regulated by guanosine triphosphate (GTP)-binding proteins, phagocytosis itself has not been considered one of these. However, previous studies have examined only unstimulated neutrophil phagocytosis. Motivated by our previous work, which showed that stimulated neutrophil phagocytosis is regulated by GTP-binding proteins (H. D. Gresham, M. G. Peters, and E. J. Brown. 1986. J. Cell Biol. 103:215a), we have examined the effect of pertussis toxin (PT) on monocyte receptor-mediated phagocytosis. PT inhibited unstimulated and fibronectin-stimulated IgG-mediated phagocytosis and also inhibited C3b-mediated phagocytosis stimulated by fibronectin or phorbol dibutyrate. Cholera toxin (CT) had no effect on unstimulated or stimulated phagocytosis mediated by IgG or C3b. PT inhibition of phagocytosis was not mediated via increases in cellular cAMP levels or by inhibition of the respiratory burst. Inhibition of phagocytosis did not result from decreased numbers of plasma membrane opsonin receptors nor decreased ability to bind opsonized targets. Although phorbol ester-stimulated phagocytosis was inhibited by PT, ligand-independent internalization of CR1 stimulated by phorbol dibutyrate proceeded normally in PT-intoxicated cells. We conclude that a PT-sensitive GTP-binding protein does regulate phagocytic function in monocytes. This protein operates on a molecular mechanism specific to the process of ingestion in both unstimulated monocytes and in cells stimulated to increase phagocytosis. Because unstimulated neutrophil phagocytosis is unaffected by PT or CT, and stimulated neutrophil phagocytosis is inhibited by both PT and CT, our data also demonstrate that monocytes and neutrophils have distinct mechanisms for regulation of phagocytic function.