Ligand-activation of the adenosine A2a receptors inhibits IL-12 production by human monocytes

Adenosine (ADO) exerts potent anti-inflammatory and immunosuppressive effects. In this paper we address the possibility that these effects are partly mediated by inhibition of the secretion of IL-12, a proinflammatory cytokine and a major inducer of Th1 responses. We demonstrate that 5'-N-ethylcarboxamidoadenosine (NECA), a nonspecific ADO analogue, and 2-p-(2-carbonyl-ethyl)phenylethylamino-5'-N-ethylcarboxamidoadenos ine (CGS-21680), a specific A2a receptor agonist, dose-dependently inhibited, in whole blood ex vivo and monocyte cultures, the production of human IL-12 induced by LPS and Stapholococcus aureus Cowan strain 1. However, the A1 receptor agonist 2-Chloro-N6-cyclopentyladenosine and the A3 receptor agonists N6-Benzyl-NECA and 1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-be ta-d -ribofuranuronamide expressed only weak inhibitory effects. On the other hand, NECA and CGS-21680 dose-dependently potentiated the production of IL-10. The differential effect of these drugs on monocyte IL-12 and IL-10 production implies that these effects are mediated by A2a receptor signaling rather than by intracellular toxicity of ADO analogue's metabolites. Moreover, CGS-21680 inhibited IL-12 production independently of endogenous IL-10 induction, because anti-IL-10 Abs failed to prevent its effect. The selective A2a antagonist 8-(3-Chlorostyryl) caffeine prevented the inhibitory effect of CGS-21680 on IL-12 production. The phosphodiesterase inhibitor Ro 20-1724 dose-dependently potentiated the inhibitory effect of CGS-21680 and, furthermore, Rp-cAMPS, a protein kinase A inhibitor, reversed the inhibitory effect of CGS-21680, implicating a cAMP/protein kinase A pathway in its action. Thus, ligand activation of A2a receptors simultaneously inhibits IL-12 and stimulates IL-10 production by human monocytes. Through this mechanism, ADO released in excess during inflammatory and ischemic conditions, or tissue injury, may contribute to selective suppression of Th1 responses and cellular immunity.     

Type V collagen selectively inhibits human endothelial cell proliferation

Type V collagen from human placenta remarkably inhibited human umbilical vein endothelial cell (HUVEC) proliferation in a dose-dependent manner when coated on the culture dishes. Other types of collagen (I, III, IV) and fibronectin enhanced HUVEC proliferation under the same conditions. The inhibitory activity of type V collagen was seen not only when it was coated on the dishes, but also when it was directly added into cell culture. The attachment effect of type V collagen did not differ from that of type I collagen. The inhibitory activity is a phenomenon selective for endothelial cells, since type V collagen did not affect the proliferation of human umbilical vein smooth muscle cells, aortic smooth muscle cells, or nasal mucosa fibroblasts.     

The nuclear IkappaB protein IkappaBNS selectively inhibits lipopolysaccharide-induced IL-6 production in macrophages of the colonic lamina propria

Macrophages play an important role in the pathogenesis of chronic colitis. However, it remains unknown how macrophages residing in the colonic lamina propria are regulated. We characterized colonic lamina proprial CD11b-positive cells (CLPMphi). CLPMphi of wild-type mice, but not IL-10-deficient mice, displayed hyporesponsiveness to TLR stimulation in terms of cytokine production and costimulatory molecule expression. We compared CLPMphi gene expression profiles of wild-type mice with IL-10-deficient mice, and identified genes that are selectively expressed in wild-type CLPMphi. These genes included nuclear IkappaB proteins such as Bcl-3 and IkappaBNS. Because Bcl-3 has been shown to specifically inhibit LPS-induced TNF-alpha production, we analyzed the role of IkappaBNS in macrophages. Lentiviral introduction of IkappaBNS resulted in impaired LPS-induced IL-6 production, but not TNF-alpha production in the murine macrophage cell line RAW264.7. IkappaBNS expression led to constitutive and intense DNA binding of NF-kappaB p50/p50 homodimers. IkappaBNS was recruited to the IL-6 promoter, but not to the TNF-alpha promoter, together with p50. Furthermore, small interference RNA-mediated reduction in IkappaBNS expression in RAW264.7 cells resulted in increased LPS-induced production of IL-6, but not TNF-alpha. Thus, IkappaBNS selectively suppresses LPS-induced IL-6 production in macrophages. This study established that nuclear IkappaB proteins differentially regulate LPS-induced inflammatory cytokine production in macrophages.     

Bacterial chaperones increase the production of soluble human TNF-alpha in Escherichia coli

Bacterial chaperones were coexpressed to enhance the production of soluble human TNF-alpha and its low-toxicity mutant in Escherichia coli. In the absence of chaperones, about 65% of wild-type TNF and 35% of mutant TNF having a deletion of N-terminal 7 amino acids and substitutions of Leu29'Ser, Ser52;Ile and Tyr56'Phe, were produced as a soluble form. In the presence of overproduced chaperones, most of wild-type and mutant TNF (>95%) were produced as a soluble form, indicating that GroEL and GroES chaperones promote folding and assembly of trimeric TNF-alpha. Bacterial chaperones could be useful in the production of TNF-alpha and its variants as well as other proteins with biological importance.     

LILRA2 selectively modulates LPS-mediated cytokine production and inhibits phagocytosis by monocytes

The activating immunoglobulin-like receptor, subfamily A, member 2 (LILRA2) is primarily expressed on the surface of cells of the innate immunity including monocytes, macrophages, neutrophils, basophils and eosinophils but not on lymphocytes and NK cells. LILRA2 cross-linking on monocytes induces pro-inflammatory cytokines while inhibiting dendritic cell differentiation and antigen presentation. A similar activating receptor, LILRA4, has been shown to modulate functions of TLR7/9 in dendritic cells. These suggest a selective immune regulatory role for LILRAs during innate immune responses. However, whether LILRA2 has functions distinct from other receptors of the innate immunity including Toll-like receptor (TLR) 4 and FcγRI remains unknown. Moreover, the effects of LILRA2 on TLR4 and FcγRI-mediated monocyte functions are not elucidated. Here, we show activation of monocytes via LILRA2 cross-linking selectively increased GM-CSF production but failed to induce IL-12 and MCP-1 production that were strongly up-regulated by LPS, suggesting functions distinct from TLR4. Interestingly, LILRA2 cross-linking on monocytes induced similar amounts of IL-6, IL-8, G-CSF and MIP-1α but lower levels of TNFα, IL-1β, IL-10 and IFNγ compared to those stimulated with LPS. Furthermore, cross-linking of LILRA2 on monocytes significantly decreased phagocytosis of IgG-coated micro-beads and serum opsonized Escherichia coli but had limited effect on phagocytosis of non-opsonized bacteria. Simultaneous co-stimulation of monocytes through LILRA2 and LPS or sequential activation of monocytes through LILRA2 followed by LPS led lower levels of TNFα, IL-1β and IL-12 production compared to LPS alone, but had additive effect on levels of IL-10 and IFNγ but not on IL-6. Interestingly, LILRA2 cross-linking on monocytes caused significant inhibition of TLR4 mRNA and protein, suggesting LILRA2-mediated suppression of LPS responses might be partly via regulation of this receptor. Taken together, we provide evidence that LILRA2-mediated activation of monocytes is significantly different to LPS and that LILRA2 selectively modulates LPS-mediated monocyte activation and FcγRI-dependent phagocytosis.     

IL-11 selectively inhibits aeroallergen-induced pulmonary eosinophilia and Th2 cytokine production

IL-11 is a pleiotropic cytokine that induces tissue remodeling with subepithelial fibrosis when expressed in the airway. Its effects on the Th2-dominated airway inflammation that is characteristic of asthma, however, are poorly understood. To characterize the effects of IL-11 on Th2 tissue inflammation, we compared the inflammatory responses elicited by OVA in sensitized mice in which IL-11 is overexpressed in a lung-specific fashion (CC10-IL-11) with that in transgene- wild-type littermate controls. Transgene- and CC10-IL-11 transgene+ mice had comparable levels of circulating Ag-specific IgE after sensitization. OVA challenge of sensitized transgene- mice caused airway and parenchymal eosinophilic inflammation, Th2 cell accumulation, and mucus hypersecretion with mucus metaplasia. Exaggerated levels of immunoreactive endothelial cell VCAM-1, mucin (Muc) 5ac gene expression and bronchoalveolar lavage and lung IL-4, IL-5, and IL-13 protein and mRNA were also noted. In contrast, OVA challenge in CC10-IL-11 animals elicited impressively lower levels of tissue and bronchoalveolar lavage inflammation, eosinophilia, and Th2 cell accumulation, and significantly lower levels of VCAM-1 and IL-4, IL-5, and IL-13 mRNA and protein. IL-11 did not cause a comparable decrease in mucus hypersecretion, Muc 5ac gene expression, or the level of expression of RANTES, monocyte chemoattractant protein-2, or monocyte chemoattractant protein-3. In addition, IL-11 did not augment IFN-gamma production demonstrating that the inhibitory effects of IL-11 were not due to a shift toward Th1 inflammation. These studies demonstrate that IL-11 selectively inhibits Ag-induced eosinophilia, Th2 inflammation, and VCAM-1 gene expression in pulmonary tissues.     

TNF-alpha inhibits flow and insulin signaling leading to NO production in aortic endothelial cells

Endothelial cells release nitric oxide(NO) acutely in response to increased "flow" or fluid shear stress(FSS), and the increase in NO production is correlated with enhancedphosphorylation and activation of endothelial nitric oxide synthase(eNOS). Both vascular endothelial growth factor and FSS activateendothelial protein kinase B (PKB) by way of incompletely understoodpathway(s), and, in turn, PKB phosphorylates eNOS at Ser-1179, causingits activation. In this study, we found that either FSS or insulinstimulated insulin receptor substrate-1 (IRS-1) tyrosine and serinephosphorylation and increased IRS-1-associated phosphatidylinositol3-kinase activity, phosphorylation of PKB Ser-473, phosphorylation ofeNOS Ser-1179, and NO production. Brief pretreatment of bovine aorticendothelial cells with tumor necrosis factor- (TNF-) inhibitedthe above described FSS- or insulin-stimulated protein phosphorylationevents and almost totally inhibited FSS- or insulin-stimulated NOproduction. These data indicate that FSS and insulin regulate eNOSphosphorylation and NO production by overlapping mechanisms. This studysuggests one potential mechanism for the development of endothelialdysfunction in disease states with alterations in insulin regulationand increased TNF- levels.

     

5'-p-Fluorosulfonylbenzoyl adenosine inhibits progesterone synthesis in human placental mitochondria

The human placental mitochondria have an ATP-diphosphohydrolase (apyrase) activity. In this paper we characterized the effect of 5'-p-fluorosulfonylbenzoyl adenosine (FSBA) on placental apyrase, and its repercussion on progesterone synthesis and oxygen consumption. Apyrase activity was inhibited by FSBA. Nucleosides tri- and diphosphates protected against FSBA inactivation, but divalent cations did not, indicating that FSBA attaches itself to an ATP-binding site of apyrase. In mitochondria, the inactivation of apyrase by FSBA was associated with inhibition of progesterone synthesis. Also, the oxygen consumption induced by ATP but not by ADP, was inhibited, clearly showing that FSBA exclusively inactivated the apyrase in human placental mitochondria. It is concluded that the apyrase activity is closely related to progesterone synthesis, probably associated with the cholesterol transport between mitochondrial membranes.     

Dengue virus selectively induces human mast cell chemokine production

Severe dengue virus infections usually occur in individuals who have preexisting anti-dengue virus antibodies. Mast cells are known to play an important role in host defense against several pathogens, but their role in viral infection has not yet been elucidated. The effects of dengue virus infection on the production of chemokines by human mast cells were examined. Elevated levels of secreted RANTES, MIP-1alpha, and MIP-1beta, but not IL-8 or ENA-78, were observed following infection of KU812 or HMC-1 human mast cell-basophil lines. In some cases a >200-fold increase in RANTES production was observed. Cord blood-derived cultured human mast cells treated with dengue virus in the presence of subneutralizing concentrations of dengue virus-specific antibody also demonstrated significantly (P < 0.05) increased RANTES production, under conditions which did not induce significant degranulation. Chemokine responses were not observed when mast cells were treated with UV-inactivated dengue virus in the presence or absence of human dengue virus-specific antibody. Neither antibody-enhanced dengue virus infection of the highly permissive U937 monocytic cell line nor adenovirus infection of mast cells induced a RANTES, MIP-1alpha, or MIP-1beta response, demonstrating a selective mast cell response to dengue virus. These results suggest a role for mast cells in the initiation of chemokine-dependent host responses to dengue virus infection.     

Amiloride selectively inhibits the urokinase-type plasminogen activator

The diuretic drug amiloride, an inhibitor of Na+ uptake, competitively inhibits the catalytic activity of the urokinase-type plasminogen activator (u-PA), with a Ki of 7 X 10(-6) M. Generation of plasmin, cleavage of peptide substrates, and interaction of u-PA with a specific macromolecular proteinase inhibitor are all prevented in the presence of the drug. In contrast, amiloride does not affect the activity of either tissue-type plasminogen activator, plasmin, plasma kallikrein or thrombin. The inhibition of u-PA by amiloride may be related to the previously reported inhibition of u-PA-type enzymes by Na+. Amiloride or related compounds could prove useful in selectively controlling u-PA-catalyzed extracellular proteolysis.     

(+)-Catechin inhibits tumour angiogenesis and regulates the production of nitric oxide and TNF-alpha in LPS-stimulated macrophages

The anti-angiogenic activity of (+)-catechin as well as its regulatory effect on the production of nitric oxide and TNFalpha were studied using in vivo and in vitro models. In vivo angiogenic activity was studied using B16F-10 melanoma cell-induced capillary formation in C57BL/6 mice. Administration of (+)-catechin significantly inhibited (36.09%) the number of tumour-directed capillaries induced by injecting B16F-10 melanoma cells on the ventral side of C57BL/6 mice. The cytokine profile in the serum of these animals showed a drastically increased level of proinflammatory cytokines such as IL-1 beta, IL-6, TNF-alpha, GM-CSF and the direct endothelial cell proliferating agent, VEGF. Administration of (+)-catechin could differentially regulate elevation of these cytokines. The differential elevation is further evidenced by the increased production of IL-2 and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the B16F-10 injected, (+)-catechin-treated animals. In vitro L929 bioassay revealed the inhibition of TNF-alpha production by (+)-catechin treatment. In the rat aortic ring assay, (+)-catechin inhibited the microvessel outgrowth at non-toxic concentrations. (+)-Catechin at non-toxic concentrations (5-25 microg/ml) showed significant inhibition in the proliferation, migration and tube formation of endothelial cells, which are the key events in the process of angiogenesis. (+)-Catechin also showed inhibitory effect on VEGF mRNA levels in B16F-10 melanoma cells. (+)-Catechin inhibited the production of NO and TNF-alpha in LPS-stimulated primary macrophages. Taken together, these results demonstrate that (+)-catechin inhibits tumour-specific angiogenesis by regulating the production of pro- and anti-angiogenic factors such as pro-inflammatory cytokines, nitric oxide, VEGF, IL-2 and TIMP-1. These results also suggest that (+)-catechin could significantly inhibit nitrite and TNF-alpha production in LPS-stimulated macrophages.     

Avarol inhibits TNF-alpha generation and NF-kappaB activation in human cells and in animal models

Avarol is a marine sesquiterpenoid hydroquinone with interesting pharmacological properties including anti-inflammatory and antipsoriatic effects. In the present study we evaluated the pharmacological effect of avarol on some inflammatory parameters related to the pathogenesis of psoriasis. Avarol inhibited tumor necrosis factor-alpha (TNF-alpha) generation in stimulated human monocytes (IC(50) 1 microM) and TNF-alpha-induced activation of nuclear factor-kappaB (NF-kappaB)-DNA binding in keratinocytes. In the mouse air pouch model, administration of avarol produced a dose-dependent reduction of TNF-alpha generation (ED(50) 9.2 nmol/pouch) as well as of interleukin (IL)-1beta, prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)) levels in pouch exudates. In the psoriasis-like model of 12-O-tetradecanoylphorbol-acetate-induced mouse epidermal hyperplasia, topical administration of avarol (0.6-1.2 micromol/site) reduced edema, myeloperoxidase activity, IL-1beta, IL-2 and eicosanoid levels in skin. Histopathological study confirmed the inhibition of epidermal hyperplasia as well as leukocyte infiltration. The reduction of cutaneous TNF-alpha by avarol was also detected by immunohistochemical analysis. Avarol was also capable of suppressing in vivo NF-kappaB nuclear translocation, determined in mouse skin. Our results suggested that antipsoriatic properties of avarol previously described could be mediated in part by the downregulation of several inflammatory biomarkers, such as TNF-alpha and NF-kappaB in psoriatic skin.     

Glutathione selectively inhibits Doxorubicin induced phosphorylation of p53Ser, caspase dependent ceramide production and apoptosis in human leukemic cells

Glutathione (GSH) is the most abundant non-protein antioxidant in mammalian cells. It has been implicated in playing an important role in different signal transduction pathways, and its depletion is an early hallmark in the progression of apoptosis in response to a number of proapoptotic stimuli. We have selectively investigated the role of GSH in cytotoxic response of Jurkat and Molt-4 human leukemic cells to the anti-cancer drug Doxorubicin. In this study, we have shown that extracellular supplementation of GSH to human leukemic cells renders them a resistant phenotype to Doxorubicin treatment. Glutathione pre-treatment inhibits Doxorubicin-induced p53Ser¹⁵ phosphorylation, caspase dependent ceramide (Cer) generation, Poly (ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation. Taken together, these results indicate that the major cellular antioxidant GSH influences the chemotherapeutic efficacy of Doxorubicin towards human leukemic cells.     

IL-9 inhibits oxidative burst and TNF-alpha release in lipopolysaccharide-stimulated human monocytes through TGF-beta

IL-9 is a Th2 cytokine that exerts pleiotropic activities on T cells, B cells, mast cells, hematopoietic progenitors, and lung epithelial cells, but no effect of this cytokine has been reported so far on mononuclear phagocytes. Human blood monocytes preincubated with IL-9 for 24 h before LPS or PMA stimulation exhibited a decreased oxidative burst, even in the presence of IFN-gamma. The inhibitory effect of IL-9 was specifically abolished by anti-hIL-9R mAb, and the presence of IL-9 receptors was demonstrated on human blood monocytes by FACS. IL-9 also down-regulated TNF-alpha and IL-10 release by LPS-stimulated monocytes. In addition, IL-9 strongly up-regulated the production of TGF-beta1 by LPS-stimulated monocytes. The suppressive effect of IL-9 on the respiratory burst and TNF-alpha production in LPS-stimulated monocytes was significantly inhibited by anti-TGF-beta1, but not by anti-IL-10Rbeta mAb. Furthermore, IL-9 inhibited LPS-induced activation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases in monocytes through a TGF-beta-mediated induction of protein phosphatase activity. In contrast, IL-4, which exerts a similar inhibitory effect on the oxidative burst and TNF-alpha release by monocytes, acts primarily through a down-regulation of LPS receptors. Thus, IL-9 deactivates LPS-stimulated blood mononuclear phagocytes, and the mechanism of inhibition involves the potentiation of TGF-beta1 production and extracellular signal-regulated kinase inhibition. These findings highlight a new target cell for IL-9 and may account for the beneficial activity of IL-9 in animal models of exaggerated inflammatory response.     

TNF-alpha inhibits adult fast myosin accumulation in myotubes

Increased muscle catabolism is frequently observed in association with inflammatory disease. TNF-alpha has been implicated as an important messenger for muscle catabolism. Experiments with cultured muscle cells exposed to TNF-alpha have produced conflicting results. In a mouse cell line (C2C12), effects ranged from catabolic to anabolic. The results reported here offer an explanation for the observed discrepancies. It was found that TNF-alpha induced proliferation of myoblasts in fully differentiated cultures in low serum media and inhibited adult fast myosin accumulation under all conditions that were tested. Furthermore, TNF-alpha caused a proliferation dependent increase in total cell protein. Addition of insulin masked the effect of TNF-alpha on total protein, but not that on adult fast myosin accumulation. Discrepancies between studies can be explained by differences in proliferation rate, the dynamic nature of C2C12 myotube cultures and accumulation of adult fast myosin. The results are consistent with a dual role of TNF-alpha: stimulation of regeneration by short-term exposure and induction of muscle wasting by prolonged exposure.