John W. Coleman

Nitric oxide in immunity and inflammation

Nitric oxide (NO) is synthesised by many cell types involved in immunity and inflammation. The principal enzyme involved is the inducible type-2 isoform of nitric oxide synthase (NOS-2), which produces high-level sustained NO synthesis. NO is important as a toxic defense molecule against infectious organisms. It also regulates the functional activity, growth and death of many immune and inflammatory cell types including macrophages, T lymphocytes, antigen-presenting cells, mast cells, neutrophils and natural killer cells. However, the role of NO in nonspecific and specific immunity in vivo and in immunologically mediated diseases and inflammation is poorly understood. NO does not act through a receptor-its target cell specificity depends on its concentration, its chemical reactivity, the vicinity of target cells and the way that target cells are programmed to respond. At high concentrations as generated by NOS-2, NO is rapidly oxidised to reactive nitrogen oxide species (RNOS) that mediate most of the immunological effects of NOS-2-derived NO. RNOS can S-nitrosate thiols to modify key signalling molecules such as kinases and transcription factors. Several key enzymes in mitochondrial respiration are also inhibited by RNOS and this leads to a depletion of ATP and cellular energy. A combination of these interactions may explain the multiple actions of NO in the regulation of immune and inflammatory cells.

5-Hydroxytryptamine Induces Mast Cell Adhesion and Migration

The neurotransmitter serotonin (5-hydroxytryptamine (5-HT)) is implicated in enhancing inflammatory reactions of skin, lung, and gastrointestinal tract. To determine whether 5-HT acts, in part, through mast cells (MC), we first established that mouse bone marrow-derived MC (mBMMC) and human CD34+-derived MC (huMC) expressed mRNA for multiple 5-HT receptors. We next determined the effect of 5-HT on mouse and human MC degranulation, adhesion, and chemotaxis. We found no evidence that 5-HT degranulates MC or modulates IgE-dependent activation. 5-HT did induce mBMMC and huMC adherence to fibronectin; and immature and mature mBMMC and huMC migration. Chemotaxis was accompanied by actin polymerization. Using receptor antagonists and pertussis toxin, we identified 5-HT1A as the principal receptor mediating the effects of 5-HT on MC. mBMMC from the 5-HT1A receptor knockout mouse (5-HT1AR−/−) did not respond to 5-HT. 5-HT did induce accumulation of MC in the dermis of 5-HT1AR+/+ mice, but not in 5-HT1AR−/− mice. These studies are the first to demonstrate an effect of 5-HT on MC. Furthermore, both mouse and human MC respond to 5-HT through the 5-HT1A receptor. Our data are consistent with the conclusion that 5-HT promotes inflammation by increasing MC at the site of tissue injury.

A Comparison of Reactive Oxygen Species Generation by Rat Peritoneal Macrophages and Mast Cells Using the Highly Sensitive Real-Time Chemiluminescent Probe Pholasin: Inhibition of Antigen-Induced Mast Cell Degranulation by Macrophage-Derived Hydrogen Peroxide

Mast cells and macrophages live in close proximity in vivo and reciprocally regulate one another’s function in various ways. Although activated macrophages possess a powerful reactive oxygen species (ROS) generating system, there is conflicting evidence regarding whether mast cells can produce ROS. We used the highly sensitive real-time chemiluminescent probe Pholasin to examine ROS release by peritoneal macrophages and mast cells isolated from OVA-sensitized rats. Macrophages stimulated with PMA (0.8 μM) or ionomycin (1 μM), but not OVA (1 μg/ml), released high-level ROS, levels of which peaked after 3–7 min and declined to baseline levels within 1 h. Superoxide was identified as the major ROS species induced by PMA but not by ionomycin. In contrast, purified mast cells stimulated with PMA released low-level ROS, which was entirely due to the contaminating (2%) macrophages, and did not release any detectable ROS in response to ionomycin or OVA at concentrations that induced degranulation. Stimulation of mixed cell populations with PMA to induce macrophage ROS release led to 50% inhibition of serotonin release from mast cells stimulated 5 min later with OVA. The PMA-induced inhibitory factor was identified as hydrogen peroxide. In conclusion, activated rat peritoneal macrophages but not mast cells produce ROS, and macrophage-derived hydrogen peroxide inhibits mast cell degranulation. The latter could be an important mechanism whereby phagocytic cells regulate mast cell activation and promote resolution of IgE-mediated inflammation.

Reciprocal effects of interleukin-4 and interferon-gamma on immunoglobulin E-mediated mast cell degranulation: a role for nitric oxide but not peroxynitrite or cyclic guanosine monophosphate.

We report that cultured rat peritoneal cells spontaneously synthesize nitric oxide and this is associated with active suppression of mast cell secretory function. Addition of interleukin‐4 (IL‐4) or the nitric oxide synthase inhibitor N‐monomethyl‐l‐arginine to peritoneal cells inhibited nitric oxide synthesis and enhanced anti‐IgE‐mediated mast cell degranulation, measured as serotonin release. Interferon‐γ (IFN‐γ) completely overcame the enhancement of serotonin release and suppression of nitrite production induced by IL‐4. Over several experiments, with or without IL‐4 and/or IFN‐γ, serotonin release correlated inversely with nitrite production. On a cell‐for‐cell basis, non‐mast cells produced ≈30 times more nitrite than mast cells in peritoneal cell populations, with or without IFN‐γ stimulation. The nitric oxide donor S‐nitrosoglutathione inhibited anti‐IgE‐induced serotonin release from purified mast cells, whereas 8‐bromo‐cyclic GMP, the guanylate cyclase inhibitor 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one, superoxide dismutase and the peroxynitrite scavenger uric acid, were without effect. We conclude that IL‐4 and IFN‐γ reciprocally regulate mast cell secretory responsiveness via control of nitric oxide synthesis by accessory cells; the nitric oxide effect on mast cells is direct but does not involve cyclic GMP or peroxynitrite.

Nitric Oxide Regulation of Human Peripheral Blood Mononuclear Cells: Critical Time Dependence and Selectivity for Cytokine versus Chemokine Expression

NO is antiproliferative for T cells and other immune cells, but there is debate over whether it influences cytokine expression and if so whether it shows cytokine selectivity. Furthermore, the NO effect may depend on exposure time. To address these issues, we precultured human PBMC with the NO donors S-nitrosoglutathione (a natural storage form of NO) or S-nitroso-N-acetyl-d-penicillamine for up to 48 h before cell activation and then monitored proliferation and cytokine and chemokine expression. S-nitrosoglutathione or S-nitroso-N-acetyl-d-penicillamine, but not their non-NO-releasing analogues, inhibited proliferation induced by PHA or IL-2, the effect declining progressively from 48 to 0 h pre-exposure to the mitogen. This was accompanied by reduced PHA-induced IL-2 release and reduced IL-2, IFN-γ, and IL-13 mRNA expression. In contrast, NO did not influence PHA-induced expression of mRNA for the chemokines lymphotactin, RANTES, IFN-γ-inducible protein, macrophage-inhibitory protein-1α, macrophage-inhibitory protein-1β, macrophage chemoattractant protein-1, and IL-8 or release of RANTES or IL-8. The NO effects were not toxic and were not accompanied by changes in PHA-induced CD25 expression. We conclude that exposure time to NO is critical to altered PBMC responsiveness and that NO inhibits expression of both Th1 and Th2 cytokines but not chemokines.

Nitric Oxide Inhibits IgE-Dependent Cytokine Production and Fos and Jun Activation in Mast Cells

NO is a cell-derived radical reported to inhibit mast cell degranulation and subsequent allergic inflammation, although whether its action is nonspecific or occurs via specific molecular mechanisms remains unknown. To examine this question, we set out to determine whether NO inhibits mast cell cytokine production, and, if so, whether it also alters FcεRI-dependent signal transduction. As hypothesized, the radical inhibited IgE/Ag-induced IL-4, IL-6, and TNF production. Although NO did not influence phosphorylated JNK, p38 MAPK, or p44/42 MAPK, it did inhibit phosphorylation of phospholipase Cγ1 and the AP-1 transcription factor protein c-Jun, but not NF-κB or CREB. NO further completely abrogated IgE/Ag-induced DNA-binding activity of the nuclear AP-1 proteins Fos and Jun. These results show that NO is capable of inhibiting FcεRI-dependent mast cell cytokine production at the level of gene regulation, and suggest too that NO may contribute to resolution of allergic inflammation.

FcεRI- and Fcγ Receptor-Mediated Production of Reactive Oxygen Species by Mast Cells Is Lipoxygenase- and Cyclooxygenase-Dependent and NADPH Oxidase-Independent

We investigated the enzymes responsible for FcεRI-dependent production of reactive oxygen species (ROS) and the influence of ROS on mast cell secretory responses. 5-Lipoxygenase (5-LO) was the primary enzyme involved in ROS production by human mast cells (huMC) and mouse bone marrow-derived mast cells (mBMMC) following FcεRI aggregation because incubation with 5-LO inhibitors (AA861, nordihydroguaiaretic acid, zileuton) but not a flavoenzyme inhibitor (diphenyleneiodonium) completely abrogated Ag-induced dichlorodihydrofluorescein (DCF) fluorescence. Furthermore, 5-LO-deficient mBMMC had greatly reduced FcεRI-dependent DCF fluorescence compared with wild type mBMMC or those lacking a functional NADPH oxidase (i.e., gp91phox- or p47phox-deficient cells). A minor role for cyclooxygenase (COX)-1 in FcεRI-dependent ROS production was demonstrated by inhibition of Ag-mediated DCF fluorescence by a COX-1 inhibitor (FR122047) and reduced DCF fluorescence in COX-1-deficient mBMMC. Complete abrogation of FcεRI-dependent ROS production in mast cells had no effect on degranulation or cytokine secretion. In response to the NADPH oxidase-stimulating agents including PMA, mBMMC and huMC produced negligible ROS. IgG-coated latex beads did stimulate ROS production in huMC, and in this experiment 5-LO and COX again appeared to be the enzymatic sources of ROS. In contrast, IgG-coated latex bead-induced ROS production in human polymorphonuclear leukocytes occurred by the NADPH oxidase pathway. Thus mBMMC and huMC generate ROS by 5-LO and COX-1 in response to FcεRI aggregation; huMC generate ROS upon exposure to IgG-coated latex beads by 5-LO and COX; and ROS appear to have no significant role in FcεRI-dependent degranulation and cytokine production.

Presentation of IFN-γ to Nitric Oxide-Producing Cells: A Novel Function for Mast Cells

We report that mast cells can bind and present IFN-γ in a functionally active form to macrophages. Flow-cytometric analysis revealed that biotinylated IFN-γ bound equally well to purified peritoneal mast cells from both IFN-γR knockout and wild-type mice, indicating a non-IFN-γR binding site. Purified peritoneal mast cells, loaded with IFN-γ for 30 min and washed, were able to induce NO synthesis by peritoneal macrophages. This response required cell contact and expression of IFN-γR on the responding macrophages, but not the mast cells. Human HMC-1 mast cells were also able to present IFN-γ to mouse macrophages. Enzyme treatment of mouse mast cells revealed that binding of IFN-γ was predominantly to chondroitin sulfate B (dermatan sulfate). Binding of IFN-γ to dermatan sulfate was confirmed by inhibition ELISA. This study demonstrates for the first time that mast cells can present IFN-γ to other cells via glycosaminoglycans. Mast cells may act as a reservoir of surface-stored functionally active cytokines.

Drug-protein conjugates—IX: Immunogenicity of captopril-protein conjugates

The immunogenicity of captopril (CP), conjugated to heterologous proteins, was investigated in male New Zealand White rabbits by monthly injections of CP-protein conjugates in Freunds Complete Adjuvant. Anti-CP antibody activity was readily detected by immunodiffusion in sera of rabbits immunized with the amide-linked CP-HSA (23:1) conjugate. Hapten inhibition studies revealed that the antigenic determinant contained CP and a lysine residue from the protein carrier. When rabbits were immunized with disulphide-linked CP-S-S-HSA (9:1) and CP-S-S-KLH (160:1) conjugates, anti-CP antibody activity was detected by a sensitive ELISA method, but not by immunodiffusion and radioligand binding assays. The specificity of the serum IgG anti-CP activity after immunization with disulphide-linked CP-S-S-protein conjugates was confirmed since anti-CP activity was inhibited by preincubation of the antisera with CP conjugated to an unrelated protein carrier (CP-S-S-OVA), but not by the corresponding unconjugated protein, nor by penicillamine-S-S-protein conjugates. These results show that disulphide-linked CP-protein conjugates are sufficiently stable to induce humoral (B lymphocyte) anti-hapten responses under the experimental conditions employed. In a separate study, delayed-type skin hypersensitivity reactions to topically applied CP were demonstrated in the guinea pig. The specific and sensitive immunochemical technique (ELISA) described here could be useful in future studies for determining whether or not patients taking CP produce antibodies to the drug.

Tumour necrosis factor‐α expression and cell recruitment in Sephadex particle‐induced lung inflammation: effects of dexamethasone and cyclosporin A

1 Tumour necrosis factor‐α (TNF‐α) is a cytokine with diverse properties consistent with a possible role in inflammatory disease. We investigated whether TNF‐α is induced during the progression of lung inflammation elicited by a particulate non‐antigenic stimulus, and whether pharmacological control of TNF‐α expression influences recruitment of specific inflammatory cell types. 2 A single intravenous injection of Sephadex particles into rats led to extensive granulomatous inflammation in lung alveolar and bronchial tissue that peaked in intensity after 24–72 h. Mononuclear cells were the principal component of granulomas, but neutrophils and eosinophils were also abundant. Numbers of mononuclear cells, neutrophils and eosinophils recovered by bronchoalveolar lavage (BAL) peaked at 72 h, 48 h and 72 h, respectively. 3 Messenger RNA encoding TNF‐α was induced in lung epithelial cells, lung granulomas and BAL cells 6 h after Sephadex administration and remained elevated for 72 h before declining to baseline by 7 days. In BAL cell populations TNF‐α protein was localized to mononuclear cells at all times points pre‐ and post‐Sephadex administration. 4 Treatment of rats with dexamethasone significantly reduced the Sephadex‐induced recruitment of mononuclear cells, neutrophils and eosinophils into the bronchoalveolar cavity, and significantly reduced TNF‐α mRNA expression by BAL cells. 5 Treatment of rats with cyclosporin A was without effect on Sephadex‐induced elevations of mononuclear cell numbers and expression of TNF‐α, but did reduce significantly recruitment of neutrophils and eosinophils to BAL cell populations. 6 These results show that a sequential asthma‐like recruitment of neutrophils, eosinophils and mononuclear cells into lung tissue can be induced by single exposure to a non‐antigenic stimulus. Pharmacological and histological studies reveal that mononuclear cell mobilization relates closely to induced TNF‐α expression, whereas mobilization of neutrophils and eosinophils appears secondary to expression of the cytokine.