Bernard Dugas

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).

Regulation of ICAM-1/CD54 expression on human endothelial cells by hydrogen peroxide involves inducible NO synthase.

Expression of the inducible isoform of nitric oxide synthase (iNOS) is stimulated by cytokines in human epithelial cells. This work indicates that incubation of human umbilical cord endothelial cells with combinations of interleukin‐1β, tumor necrosis factor α, and interferon‐γ stimulated the synthesis of iNOS mRNA, as detected by reverse transcriptase‐polymerase chain reaction. It is important to note that 50, 100, and 200 μM hydrogen peroxide was able to stimulate iNOS directly. Furthermore, 100 μM H2O2 enhanced synthesis of the oxidation products, nitrite (NO) and nitrate (NO) at 12 and 36 h. iNOS protein, detected by Western blot analysis, as well as L‐citrulline levels, were also increased. When endothelial cell monolayers were incubated for 1 h with 100 μM H2O2 and subsequently with cytokines, iNOS mRNA was further augmented. Under the same conditions, we regularly observed an inhibition (25%) of intercellular adhesion molecule‐1 (ICAM‐1/CD54) expression. The latter was reversed when the NOS inhibitor NG ‐monomethyl‐L‐arginine was added, as shown by flow cytometry. These data suggest a specific effect of endogenous hydroperoxides on the biosynthesis and processing of the human endothelial iNOS isoform. We propose that H2O2 induces a temporary NO‐dependent modulation of adhesion molecule expression to limit the tissue destruction that accompanies the vascular recruitment of leukocytes. J. Leukoc. Biol. 67: 327–334; 2000.

Functional interaction between β2-adrenoceptor agonists and interleukin-4 in the regulation of CD23 expression and release and IgE production in human

Normal human peripheral blood mononuclear cells (PBMC) produced IgE when stimulated with IL-4. In the present report it was shown that beta 2-adrenoceptor agonists, salbutamol and fenoterol, potentiated the IL-4-induced IgE production without significantly affecting the expression of the low affinity receptor for IgE at the cell surface of monocytes and B lymphocytes. However, beta 2-adrenoceptor agonists were shown to enhance at day 7 the IL-4-induced release of the soluble form of CD23 (sCD23) by PBMC. This effect was specific since a beta-adrenoceptor antagonist, D,L-propranolol, inhibited the IL-4-induced IgE production by these cells. Alternatively, the beta 2-adrenoceptor agonists inhibited the production by these cells of interferon-gamma (IFN-gamma) but did not affect the production of IL-4 when stimulated with phytohemagglutinin A + a phorbol ester. These data suggest that beta 2-adrenoceptor agonists influence the IL-4-induced IgE production in humans by enhancing the release of sCD23 and inhibiting the production of endogenous IFN-gamma. In addition to the effect on the IL-4-induced IgE production it was shown that beta 2-adrenoceptor agonists potentiated the effect of IL-4 on a human promonocytic cell line, U 937, by enhancing CD23 expression and release and by inducing the differentiation of these cells into monocyte-like cells. Taken together, these data indicate that beta 2-adrenoceptor agonists potentiated the effect of IL-4 and that this functional interaction is different considering the cell-lineage and the stage of differentiation of these cells.

Contribution of Nitric Oxide to the Apoptotic Process in Human B Cell Chronic Lymphocytic Leukaemia

B cell chronic lymphocytic leukaemia (B-CLL) is characterised by defective apoptosis that cannot be explained solely on the basis of the known chromosomal abnormalities. We and other have now reported that the leukemic cells spontaneously display the inducible isoform of nitric oxide synthase, iNOS. Inhibition of the iNOS pathway leads to increased apoptosis of the tumoral cells in vitro, indicating that the endogenous release of NO contributes to their resistance to the normal apoptotic process. The factors that induce the expression of iNOS in vivo in the leukemic cells are not yet identified. Yet, as interaction of B-CLL leukemic cells with bone marrow stromal cells promotes their survival, the involvement of adhesion molecules and integrins may be suspected. The engagement of CD23 stimulates iNOS activation in the tumoral cells, suggesting that in vivo interaction of CD23 with one of its recognised lig-ands may contribute to iNOS induction. A role for CD40-CD40 ligand interaction may also be hypothesised. The mechanisms involved in the anti-apoptotic role of NO are not fully understood, but may implicate the inhibition of caspase activity, hence the impairment of the Fas pathway. In addition, the mitochondrial membrane potential disruption appears to be a NO-sensitive step in the apoptosis cascade. The presence of a NOS displaying anti-apoptotic properties has now been recognised in different cell types, including various leukaemia. A better knowledge of the mechanisms governing the ultimate fate of NO, anti-versus pro-apoptotic would allow the development of new therapeutic approaches for the treatment of these diseases.

Modulation by interleukin-4 of cytokine release from mononuclear phagocytes in asthma

BACKGROUND Interleukin (IL)-4 is involved in IgE upregulation and downregulates cytokine release by mononuclear phagocytes. Mononuclear cells release greater amounts of IL-1, tumor necrosis factor-alpha, and IL-6 in patients with asthma than in control subjects, but the effect of IL-4 on cells from patients with asthma is unknown. The effects of IL-4 on the release of IL-1, tumor necrosis factor-alpha, and IL-6 by monocytes and alveolar macrophages were compared in 19 patients with asthma and 18 control subjects. METHODS The release of IL-1, tumor necrosis factor-alpha, and IL-6 from unstimulated and lipopolysaccharide stimulated monocytes and alveolar macrophages was measured by ELISA. The effect of 30 U of IL-4 on the release of these cytokines was studied. RESULTS Lipopolysaccharide-stimulated monocytes released significantly fewer cytokines in patients with asthma than in control subjects. IL-4 significantly inhibited cytokine release by monocytes of both groups. Unstimulated alveolar macrophages from patients with asthma released more cytokines than those of control subjects. Lipopolysaccharide induced a significantly greater increase in cytokine release in alveolar macrophages of control subjects in comparison with asthmatic subjects. IL-4 abolished the release of cytokines in alveolar macrophages from control subjects and had a minimal inhibitory effect on alveolar macrophages from patients with asthma. CONCLUSIONS Alveolar macrophages from patients with asthma are hyperreactive but less prone to lipopolysaccharide stimulation and IL-4-downregulation than those from normal subjects.

Nitric Oxide Production in Human Macrophagic Cells Phagocytizing Opsonized Zymosan: Direct Characterization by Measurement of the Luminol Dependent Chemilurninescence

When differentiated into mature macrophages by the combination of all-trans retinoic acid and 1, 25-dihy-droxyvitamin D3, the human promonocytic cell lines U937 and THP-1 expressed inducible nitric oxide syn-thase (iNOS) transcripts. During their differentiation, the cells acquired the capacity to produce not only superoxide anion (O2−) but also nitric oxide (NO) in response to IgG (or IgE)-opsonized zymosan. The inhibitors of the iNOS pathway, aminoguanidine and NG-monomethyl-L-arginine (L-NMMA), suppressed the production of .NO and enhanced the steady-state concentration of O2− determined. Conversely, super-oxide dismutase (SOD) scavenged the O2− released and increased the NO-derived nitrite concentration detected. These data suggested a possible interaction between O2− and NO. In differentiated U937 (or THP-1) cells, IgG or IgE-opsonized zymosan induced a strong time-dependent luminol-dependent chemiluminescence (LDCL), which was abroated by SOD and partially inhibited by aminoguanidine or L-NMMA. Sinc...

Beta 2-adrenoceptor agonists regulate the IL-4-induced phenotypical changes and IgE-dependent functions in normal human monocytes.

The β 2‐adrenoceptor agonists salbutamol and fenoterol were tested for their regulatory effects on human monocyte phenotype and functions, either alone or in combination with interleukin‐4 (IL‐4). These drugs enhanced in a dose‐dependent manner the IL‐4‐induced membrane and mRNA expression of the low‐affinity receptor for immunoglobulin E (IgE) (CD23), as well as the release of its soluble form, sCD23. Salbutamol and fenoterol alone elicited expression of the monomorphic β 2‐chain (CD18) of the leukocyte functional antigen (LEA1) family. This effect appeared to be restricted to CD11b (CR3) and CD11c (gp 150–95), because CD11a (LFA‐1α chain) was not modified, β 2‐Adrenoceptor stimulation was also found to potentiate the effect of IL‐4 on CD11b, CD11c, and CD18 expression. In contrast, these agents alone did not alter the level of major histocompatibility complex class II and CD14 antigens or modify their respective up‐ and down‐regulation by IL‐4. Ligation of CD23 on IL‐4‐preincubated (CD23*) monocytes with IgE/anti‐IgE immune complexes induced the release of free radicals nitric oxide and of the proinflammatory mediators IL‐6 and thromboxane B2 (TxB2). Addition of salbutamol, inactive alone, potentiated the generation of superoxide anion and of nitric oxide generation, as well as the production of IL‐6 and TxB2 triggered by CD23 ligation. These results indicate that β 2‐adrenoceptor stimulation potentiates in vitro the IL‐4‐induced phenotypical and functional changes on monocytes and suggest that such an interaction could occur in IgE‐dependent immune reactions. J. Leukoc. Biol. 55: 313–320; 1994.

Pharmacological Modulation of the Antigen-Induced Expression of the Low-Affinity IgE Receptor (FcεRII/CD23) on Rat Alveolar Macrophages

Brown-Norway (BN) rats were sensitized by 3 aerosol exposures to ovalbumin (OA; 10 mg/ml) at days 1, 3 and 14. At day 21, the rats were challenged with the antigen or vehicle by aerosol. Alveolar macrophages (AM) were obtained by bronchoalveolar lavage and the expression of Fc epsilon RII/CD23 was assessed by flow cytometry after staining with the BB10 monoclonal antibody. A maximum of 74% of the AM from sensitized and challenged BN rats expressed FC epsilon RII/CD23 24 h after OA exposure, compared to 12% of the cells from rats exposed to vehicle. Sprague-Dawley rats were passively sensitized by intravenous injection of 0.1 or 0.05 ml/kg mouse ascitic fluid containing dinitrophenyl (DNP)-specific monoclonal IgE (2682-1) and after 24 h exposed to an aerosol of 5 mg/ml of DNP-bovine serum albumin for 30 min. In this case also, antigen exposure induced the expression of Fc epsilon RII/CD23 on 75% AM, compared to 17% AM from saline-challenged rats. Such an induction of Fc epsilon RII/CD23 on AM was, however, not observed when the animals were challenged with either histamine, serotonin or acetylcholine by aerosol. The antigen-induced expression of Fc epsilon RII/CD23 on AM was inhibited upon treatment of the rats with ketotifen or beclomethasone. In addition, oral or aerosol administration of respectively BN 50730 or BN 52021 (two structurally unrelated platelet-activating factor antagonists), inhibited the antigen-induced Fc epsilon RII/CD23 expression on AM, indicating the participation of this lipid mediator in this process.

Beta-2-Adrenoceptor Agonists Up-Regulate the in vitro FcEpsilon Receptor II/CD23 Expression on, and Release from, the Promonocytic Cell Line U937 and Human Blood Monocytes

The possible regulatory role of beta 2-adrenoceptor agonists in the modulation of CD23 on the human promonocytic cell line U937 and on human monocytes was investigated. Incubation for 48 h in the presence of interleukin-4 (IL-4; 30 U/ml) induced optimal expression and release of CD23 on both U937 cells and human monocytes. When a beta 2-adrenoceptor agonist, either salbutamol or fenoterol, was added to U937 cells or monocytes both the IL-4-induced CD23 expression and release were markedly up-regulated. This effect was dose-dependent, starting at 10 nM and reaching a maximum at 1 microM final concentration of either salbutamol or fenoterol. The potentiating effect of salbutamol and fenoterol on CD23 expression and release was not observed when a beta-adrenoceptor antagonist, either D,L-propranolol (1 microM) or butoxamine (1 microM), was added to the incubation medium. The alpha-adrenoceptor agonist norepinephrine (1 microM) was ineffective in enhancing the IL-4-induced expression and release of CD23 from U937 cells or human monocytes, demonstrating the specificity of the beta 2-adrenoceptor-mediated effect. In the absence of IL-4, a moderate but significant increase in the CD23 expression on U937 cells and human monocytes by these drugs was observed, as compared to the basal values. These results indicate that, besides their bronchodilator effect, beta 2-adrenoceptor agonists may regulate IgE-dependent processes.

Differential pattern in circulating nitrogen derivatives, lactoferrin, and anti-lactoferrin antibodies in HIV type 1 and HIV type 2 infections

HIV-1 infection is associated with a dramatic reduction in antioxidative molecules both at the cellular level and in the circulation. This is particularly so for lactoferrin, an iron-binding protein involved in natural defenses (antimicrobial and antiviral activities, etc.) and found in whole secretions, including milk and mucus. In addition to its ability to chelate iron ions, lactoferrin inhibits hydroxy radical formation and interacts with nitric oxide (NO). Levels of plasma lactoferrin decreased in HIV-1-infected patients in correlation with progression of the disease, and highly specific anti-lactoferrin autoantibodies increased. This profile was specific to HIV-1 infection; it was not found in HIV-2-infected patients. In parallel with the drop in lactoferrin, a marked increase in circulating nitrogen derivatives was observed in HIV-1-infected patients, whereas low levels were found in normal donors and in HIV-2-infected patients. These data suggested hyperstimulation of the NO pathway throughout HIV-1 but not HIV-2 infection. This overproduction of NO could play an important role in the development of AIDS symptoms and signs.