Grape polyphenols and propolis mixture inhibits inflammatory mediator release from human leukocytes and reduces clinical scores in experimental arthritis.

Polyphenols from red fruits and bee-derived propolis (PR) are bioactive natural products in various in vitro and in vivo models. The present study shows that hematotoxicity-free doses of grape polyphenols (GPE) and PR differentially decreased the secretion of pro-inflammatory cytokines from activated human peripheral blood leucocytes. While GPE inhibited the monocytes/macrophage response, propolis decreased both monokines and interferon γ (IFNγ) production. When used together, their distinct effects lead to the attenuation of all inflammatory mediators, as supported by a significant modulation of the transcriptomic profile of pro-inflammatory genes in human leukocytes. To enforce in vitro data, GPE+PR were tested for their ability to improve clinical scores and cachexia in chronic rat adjuvant-induced arthritis (AA). Extracts significantly reduced arthritis scores and cachexia, and this effect was more significant in animals receiving continuous low doses compared to those receiving five different high doses. Animals treated daily had significantly better clinical scores than corticoid-treated rats. Together, these findings indicate that the GPE+PR combination induces potent anti-inflammatory activity due to their complementary immune cell modulation.

Malvidin-3-O-ß glucoside, major grape anthocyanin, inhibits human macrophage-derived inflammatory mediators and decreases clinical scores in arthritic rats.

Polyphenolic anthocyanins are major colorful compounds in red fruits, known to prevent cardiovascular and other diseases. Grape polyphenols are a mixture of various molecules and their exact contribution to above bioactivities remains to be clarified. In the present study, we first analyzed the effect of purified grape-derived compounds on human peripheral blood mononuclear cell (PBMC) survival, proliferation, as well as for their ability to inhibit the activation of human normal macrophages. Data indicated that malvidin-3-O-ß glucoside (Malßg), the major grape anthocyanin, is bioactive with no toxicity on human PBMC. Malßg decreased the transcription of genes encoding inflammatory mediators, confirmed by the inhibition of TNFα, IL1, IL-6 and iNOS-derived nitric oxide (NO) secretion from activated macrophages. As Malßg also inhibited inflammatory response of rat macrophages, we investigated the anti-inflammatory potential of Malßg in chronic rat adjuvant-induced arthritis (AIA). Malßg significantly diminished inflammatory cachexia and arthritic paw scores in AIA rats at both therapeutic and preventive levels. In vivo effects of Malßg correlated with down-regulation of NO generation from AIA rats' peritoneal macrophages ex vivo. These data indicate that Malßg, major grape anthocyanin, is a potent anti-inflammatory agent in vitro and in vivo, without detectable toxic effect.

Nitric oxide induces the apoptosis of human BCR-ABL-positive myeloid leukemia cells: evidence for the chelation of intracellular iron.

Anti-leukemia activity of human macrophages involves the generation of nitric oxide (NO) derivatives. However, leukemic transformation may involve mechanisms that rescue cells from NO-mediated apoptosis. In the present work, we analyzed the effects of exogenous NO on the proliferation of BCR-ABL(+) chronic myelogenous leukemia (CML) cells. As normal leukocytes, the proliferation of leukemia cells was inhibited by SNAP (S-nitroso-N-acetyl-penicillamine), GEA (Oxatriazolium amino-chloride), and SIN-1 (Morpholino-sydnonimine), whereas SNP (sodium nitroprusside) had no effect on leukemia cell growth. SIN-1 induced higher anti-proliferation activity in BCR-ABL(+) cells, compared to normal hemopoietic cells. Inhibition of leukemia cell proliferation correlated with increased apoptosis and DEVDase activity. The simultaneous addition of exogenous iron reversed NO-mediated inhibition of cell growth, caspase activation and apoptosis in all BCR-ABL(+) cells tested. The quantification of intracellular iron levels in leukemia cells indicated that NO induced an early, dose-dependent decrease in ferric iron levels. Accordingly, elevation of intracellular iron protected leukemia cells from NO-mediated apoptosis. Together, the present work reveals the presence of an iron-dependant mechanism for leukemia cell rescue from NO-induced growth inhibition and apoptosis.

Role of iron in tumor cell protection from the pro-apoptotic effect of nitric oxide.

F2The host defense against tumor cells is in part based upon the production of nitric oxide (NO) by activated macrophages. However, carcinogenesis may involve mechanisms that protect tumor cells from NO-mediated apoptosis. In the present study, we have assessed the effects of exogenous NO on the proliferation and survival of human liver (AKN-1), lung (A549), skin (HaCat), and pancreatic (Capan-2) tumor cell lines, compared with normal skin-derived epithelial cell cultures. Except to the HaCat cell line, all of the other human epithelioid cells were sensitive to the antiproliferation effect of S-nitroso-N-acetyl-penicillamine or Deta NONOate, whereas tumor cells had low if any response to sodium nitroprusside. Growth inhibition with exogenous NO correlated with increased apoptosis, but was not mediated by cyclic GMP, peroxynitrite generation, or poly(ADP-ribose) polymerase modulation, all of which involved in NO-mediated growth inhibition of normal skin-derived epithelial cell cultures. The simultaneous addition of iron-containing compounds protected tumor cells from NO-mediated growth inhibition and apoptosis. Intracellular iron quantification indicated that, as deferoxamine, exogenous NO significantly decreased intracellular ferric iron levels in tumor cells. Together, the current study reveals that intracellular iron elevation rescues tumor cells from NO-mediated iron depletion and subsequent growth inhibition and apoptosis.

Differential effect of interferon alpha on chronic myelogenous leukaemia and normal haematopoietic progenitors in a stromal cell co-culture context: role of the flt3 ligand.

Interferon alpha (IFN-alpha) is used to treat chronic myelogenous leukaemia (CML) patients. However, its target(s) remain(s) unknown. One possibility is that there is a differing sensitivity of the leukaemic from the normal colony-forming cell (CFC) compartments to IFN-alpha. Co-cultures of progenitors with stromal cells provide a valuable tool to dissect direct and indirect activities of IFN-alpha. In this study, we have used endothelial cells (EC) as a source of stromal cells. In co-cultures of normal progenitors with EC, IFN-alpha increased the generation of clonogenic cells, mainly via an increased production of flt3 ligand (FL) by EC. In contrast, in co-cultures of CML progenitors with EC, IFN-alpha inhibited the generation of clonogenic cells, mainly by direct inhibition on the progenitors, the up-regulation of FL production by stromal cells being unable to compensate for the direct inhibitory effects of IFN-alpha. These data provide evidence for a differential effect of IFN-alpha on the growth of CML and normal CFC cells in a stromal context and suggest that an alteration in the response of CML progenitor cells to FL is important in the explanation of this differential effect.

The human immune response during cutaneous leishmaniasis: NO problem.

During some helminth infections, increased expression of the low-affinity receptor for IgE (CD23/FcepsilonRII) by macrophages and/or increased levels of plasma IgE have been seen, but their role in host protection or disease progression remains unclear. Recently, crosslinking of CD23 was shown to promote intracellular killing of Leishmania parasites in human macrophages, a phenomenon involving the production of tumor necrosis factor alpha and nitric oxide (NO). Based upon various in vitro and in vivo studies of human cutaneous leishmaniasis, Djavad Mossalayi, Michel Arock, Dominique Mazier, Philipe Vincendeau and Ioannis Vouldoukis here propose a model for an immune response that involves CD23-IgE-mediated NO release during protection, as well as during active cutaneous leishmaniasis.

Critical role of nitric oxide during the apoptosis of peripheral blood leukocytes from patients with AIDS.

BACKGROUND: Highly active antiretroviral therapies (HAART) increase the CD4(+) cell count, but complete normalization of this parameter has not been obtained in some patients. As oxidative stress plays an important role during human immunodeficiency virus type 1 (HIV-1)-associated dementia and lymphocyte apoptosis, we asked whether the nitric oxide (NO) pathway plays a role in the in vitro survival of peripheral blood mononuclear cells (PBMC) from HIV-1(+) patients and how it correlates with peripheral CD4(+) cell levels. MATERIALS AND METHODS: PBMC were isolated from patients with AIDS and assayed for apoptosis and proliferation in the presence of various chemicals, including agonists or antagonists of the NO pathway. Data were then compared with several in vivo parameters from the same patients. RESULTS: Apoptosis of PBMC in the presence of exogenous NO is significantly higher in patients with low peripheral CD4(+) cell levels than in patients with high CD4(+) cell numbers or seronegative individuals. In addition, endogenous NO inhibition rescues cells from apoptosis in AIDS patients with low circulating CD4(+) cell numbers and helps recovery of the T cell proliferative response. NO-mediated apoptosis does not require cGMP but involves peroxynitrite generation, PARP activation, and NAD(+) depletion. CONCLUSIONS: Taken together, the data suggest the involvement of NO during the apoptosis and functional impairment of lymphocytes in patients with AIDS.

Inducible nitric oxide synthase and proinflammatory cytokine expression by human keratinocytes during acute urticaria.

BACKGROUND: IgE/allergen-dependent activation of skin mast cells is involved in acute urticaria and leads to their IL-4 release. Previously we have demonstrated in vitro the induction of the low-affinity receptor for IgE (CD23/Fc epsilon RII) in human keratinocytes (HK) upon stimulation with IL-4. In addition, we have observed that ligation of CD23 on keratinocytes induced type II nitric oxide synthase (iNOS), leading to the release of nitric oxide (NO) and proinflammatory cytokines (TNF-alpha, IL-6). According to these in vitro data, we explored whether keratinocytes could also express iNOS, TNF-alpha, IL-6, and CD23 in acute urticaria, an in vivo model in which activation of mast cells by IgE/allergen immune complexes is involved. MATERIALS AND METHODS: INOS, TNF-alpha, IL-6, and CD23 expression by keratinocytes was studied in acute urticaria (n = 11) in biopsies from lesional and autologous normal skin by immunohistochemistry, in situ hybridization, or RT-PCR. Nitrites and TNF-alpha synthesis were assayed in supernatants of cultured lesional keratinocytes. RESULTS: INOS mRNA expression was demonstrated with RT-PCR in 10 biopsies out of 11 sections of acute urticaria lesional skin. Immunohistochemistry showed that this iNOS positivity originated from keratinocytes located close to the dermoepidermal junction; TNF-alpha and IL-6 mRNA transcription was observed in all but one iNOS+ biopsy. Immunostaining and in situ hybridization with CD23-specific probes were strong in all but one iNOS+ skin biopsy. Noninflamed autologous skin was negative for iNOS (except for a weak positivity in one case), cytokines, and CD23. CONCLUSION: The colocalization of iNOS, proinflammatory cytokines, and CD23 within keratinocytes in acute urticaria demonstrates that these cells play an important role in the initiation and maintenance of the inflammatory reaction during this disease in humans through activation of the iNOS pathway by CD23 ligation with IgE/allergen immune complexes.

Interleukin-10 and interleukin-4 inhibit intracellular killing of Leishmania infantum and Leishmania major by human macrophages by decreasing nitric oxide generation.

The host response to Leishmania infection is regulated by a specific pattern of local cytokine production. We investigated the effect of interleukin (IL)-10 and IL-4 on the leishmanicidal activity of human macrophages (M phi). As with L. major, intracellular killing of L. infantum by human M phi was obtained following ligation of surface CD23 or cell treatment with interferon-gamma (IFN-gamma). This leishmanicidal activity required nitric oxide (NO) generation by activated M phi, and it was partially mimicked by cell treatment with chemical NO donors. Addition of recombinant human IL-10 or IL-4 to CD23 mAb or IFN-gamma decreased L. infantum and L. major killing by infected M phi. IL-10 was more potent than IL-4 in inhibiting the leishmanicidal activity of human M phi. Inhibition of Leishmania killing by IL-4 and IL-10 correlated with decreased NO generation from M phi, and was reversed when exogenous NO was added to cell cultures. Therefore, IL-10 and IL-4 down-regulate leishmanicidal activity of human M phi, in part by inhibiting NO generation by these cells.

CD23/Fc epsilon RII: signaling and clinical implication.

CD23 is an activation antigen expressed by various human hematopoietic cells, tissular epithelial cells and represents the major low affinity receptor for IgE (Fc epsilon RII). In its membrane and soluble forms, CD23 has multiple ligands that enable this molecule to trigger various functions in human and murine cells. In this issue, we discussed the intracellular signaling events induced by soluble CD23 and the ligand involved in each target cell. Signal transduction through surface CD23 ligation is linked to cyclic nucleotides and nitric oxide (NO) pathways in various human cells and in rat macrophages. Recent in vivo data suggest a regulatory role for these signals during various human physiopathological situations such as hemopoiesis, anti-tumoral defense, inflammation, allergy, microbicidal activity of macrophages and eosinophils, skin disease, and HIV infection.

Induction of IL-10 synthesis by human keratinocytes through CD23 ligation: a cyclic adenosine 3',5'-monophosphate-dependent mechanism.

Ligation of the low affinity receptor for IgE, CD23/Fc epsilonRII, in human keratinocytes (HK) and monocytes induces the synthesis of proinflammatory cytokines (IL-6 and TNF-alpha), partly under the dependence of cAMP and nitric oxide pathways. Moreover, CD23 ligation induces IL-10 production in human monocytes. Since synthesis of IL-10 by HK is still a matter of debate, we investigate whether keratinocytes could produce IL-10 upon CD23 stimulation. Here, our data show that CD23 ligation induces significant IL-10 synthesis in HK, a phenomenon inhibited by cAMP antagonists, but not by inhibitors of the nitric oxide pathway. Accordingly, cAMP agonist induced significant IL-10 synthesis by HK, while nitric oxide-releasing chemical did not. Treatment of HK with anti-IL-10 mAb potentiated their CD23-mediated TNF-alpha synthesis. These data indicate that engagement of surface CD23 on human keratinocytes induces the synthesis of IL-10, which, in turn, down-regulates their proinflammatory response.

Evidence for direct interaction between mast cells and Leishmania parasites.

When stimulated through IgE-(or IgG-) immune complexes with parasite antigens, mast cells can release several cytokines, including IL-4, IL-6, IL-10, IL-12, Interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) that may influence the host response to Leishmania major in modulating lesion size and persistence during experimental infection in the mouse. Moreover, recent data demonstrated that mast cells are able to be antibody-independently activated by direct contact with bacteria, making them important elements in innate immunity. Given these data, we asked whether cell-parasite contact could directly induce mast cell mediator release and whether mast cells could be infected by L. major or L. infantum parasites. In this study, we showed that a pure homogeneous population of mouse bone marrow derived mast cells (BMMC) in contact with living L. major or L. infantum promastigotes, but not with attenuated parasites or soluble parasite antigens, released preformed mediators such as beta-hexosaminidase and the preformed pool of TNF-alpha within minutes. Furthermore, direct cell-parasite contact induced TNF-alpha synthesis by mast cells within hours. Moreover, we demonstrated by in vitro co-culture experiments that metacyclic L. major or L. infantum promastigotes are directly infective for a significant proportion of BMMC and are transformed into intracellular amastigotes. Taken together, these data suggest that mast cell can participate in the first line of defence, i.e. innate immunity, during local cutaneous infection with Leishmania parasites.

Role of nitric oxide in the anti-tumoral effect of retinoic acid and 1,25-dihydroxyvitamin D3 on human promonocytic leukemic cells.

All trans retinoic acid and vitamin D3 derivatives are well known for their antileukemic activity, while the precise mechanism of this effect remains to be clarified. Using human leukemic U937 and THP-1 promonocytic cell lines, we analyzed the effect of all-trans retinoic acid (RA) and/or 1,25-dihydroxyvitamin D3 (VD) on the generation of nitric oxide (NO), a potent antitumoral mediator. U937 cell differentiation with VD or with both RA and VD (RA/VD) correlated with gene transcription and functional expression of inducible nitric oxide synthase (iNOS). Nitrites and L-citrulline were also detected in U937 cell supernatants as soon as 24 hours following cell incubation with VD or RA/VD, but not in cells treated with RA alone. Inhibition of iNOS activity by NG-monomethyl-L-arginine (LNMMA) significantly decreased in vitro U937 cell differentiation with VD and RA/VD as shown by the expression of cell differentiation markers (CD14 and CD68) and by the capacity of these cells to undergo a luminol-dependent chemiluminescence in response to opsonized zymosan. Similar results were obtained using the THP-1 cell line strengthening the role of NO in the VD- and RA/VD-induced growth arrest and terminal differentiation of promonocytic leukemia cells.

Theophylline synergizes with chlorambucil in inducing apoptosis of B-chronic lymphocytic leukemia cells.

We tested the effects of theophylline, a phosphodiesterase inhibitor inducing intracellular accumulation of cyclic adenosine monophosphate (cAMP), on malignant B cells from 15 patients with B-chronic lymphocytic leukemia (B-CLL). We observed a large increase in apoptotic cell numbers (mean, 90% v 20% in medium alone) in the presence of theophylline (100 micrograms/mL) or chlorambucil (10 mumol/L) after 72 hours of incubation. Maximal apoptosis (90%) was reached after 36 hours when the two drugs were used together at fourfold lower concentrations, indicating a synergistic effect; no effect was observed with normal B cells, suggesting that the combination might have therapeutic interest. Chlorambucil induced intracellular Ca+2 influx, pointing to the involvement of two signaling pathways that might explain its synergy with theophylline through their effects on oncogenes. The expression of bcl-2 protein, a proto-oncogene inhibiting apoptosis, decreased after incubation with the drugs, while c-myc, recently described as having a potent role in apoptosis, was overexpressed. For p53 we observed an overexpression in the presence of chlorambucil or both theophylline-chlorambucil and a decrease after theophylline incubation. Chlorambucil- and theophylline-induced apoptosis was partially inhibited by interleukin-4 (IL-4), which also abrogated the effects on oncogene expression. These results provide insight into the mechanisms underlying B-CLL apoptosis and suggest that the theophylline-chlorambucil combination may be of therapeutic value in this setting.

CD23-mediated nitric oxide synthase pathway induction in human keratinocytes is inhibited by retinoic acid derivatives.

Retinoids exert various functions including anti-proliferative and anti-inflammatory effects on many cell types including keratinocytes and are widely used in skin diseases, such as psoriasis and acne. We have previously shown that human keratinocytes express low affinity immunoglobulin E receptor (FcepsilonRII/CD23) when stimulated with interleukin-4. Immunoglobulin E ligates CD23 and induces the production of nitrites (reflecting the mobilization of the nitric oxide [NO]-pathway) and tumor necrosis factor-alpha by human keratinocytes. Here, 13-cis and all-trans retinoic acid (RA) were shown to reduce the production of nitrites by immunoglobulin E-activated keratinocytes by 80% in a time- and concentration-dependent fashion. As a consequence, RA derivatives also reduced the production of tumor necrosis factor alpha by these cells by 70%. The level of inducible NO synthase activity in activated human keratinocytes was significantly decreased upon treatment of the cells with RA derivatives (inhibition by 60% of the mean inducible NO synthase activity with 13-cis RA, 2 microM). Treatment for 24 h with RA derivatives almost completely abolished transcription of inducible NO synthase-specific mRNA in activated keratinocytes. Therefore, RA derivatives downregulate tumor necrosis factor-alpha release and the NO-transduction pathway through the inhibition of inducible NO synthase transcription. Together, our data provide evidence for inhibition of the NO-pathway by 13-cis and all-trans retinoic acid on CD23-activated human keratinocytes. These data may clarify the mechanism of the anti-inflammatory activity of RA derivatives in skin diseases.

Role of IgE immune complexes in the regulation of HIV-1 replication and increased cell death of infected U1 monocytes: involvement of CD23/Fc epsilon RII-mediated nitric oxide and cyclic AMP pathways.

BACKGROUND: IgE/anti-IgE immune complexes (IgE-IC) induce the release of multiple mediators from monocytes/macrophages and the monocytic cell line U937 following the ligation of the low-affinity Fc epsilon receptors (Fc epsilon RII/CD23). These effects are mediated through an accumulation of cAMP and the generation of L-arginine-dependent nitric oxide (NO). Since high IgE levels predict more rapid progression to acquired immunodeficiency syndrome, we attempted to define the effects of IgE-IC on human immunodeficiency virus (HIV) production in monocytes. MATERIALS AND METHODS: Two variants of HIV-1 chronically infected monocytic U1 cells were stimulated with IgE-IC and virus replication was quantified. NO and cAMP involvement was tested through the use of agonistic and antagonistic chemicals of these two pathways. RESULTS: IgE-IC induced p24 production by U1 cells with low-level constitutive expression of HIV-1 mRNAs and extracellular HIV capsid protein p24 levels (U1low), upon their pretreatment with interleukin 4 (IL-4) or IL-13. This effect was due to the crosslinking of CD23, as it was reversed by blocking the IgE binding site on CD23. The IgE-IC effect could also be mimicked by crosslinking of CD23 by a specific monoclonal antibody. p24 induction by IgE-IC was then shown to be due to CD23-mediated stimulation of cAMP, NO, and tumor necrosis factor alpha (TNF alpha) generation. In another variant of U1 cells with > 1 log higher constitutive production of p24 levels (U1high), IgE-IC addition dramatically decreased all cell functions tested and accelerated cell death. This phenomenon was reversed by blocking the nitric oxide generation. CONCLUSIONS: These data point out a regulatory role of IgE-IC on HIV-1 production in monocytic cells, through CD23-mediated stimulation of cAMP and NO pathways. IgE-IC can also stimulate increased cell death in high HIV producing cells through the NO pathway.

Nitric oxide production by human monocytes: evidence for a role of CD23.

Nitric oxide (NO) appears to be an important and pleiotropic bioregulator of immune responses. The existence of the NO synthase (NOS) pathway in human monocytes/macrophages remains a subject of controversy, despite an increasing number of reports suggesting that human monocytes produce NO in vitro in response to various stimuli. Here, Bernard Dugas and colleagues consider the arguments supporting these conclusions, with particular emphasis on the results obtained by ligation of the low-affinity IgE receptor (Fcepsilon RIIb/CD23b).