SUMMARYWe 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--arginine to peritoneal cells inhibited nitric oxide synthesis and enhanced anti-IgE-mediated mast cell degranulation, measured as serotonin release. Interferon-c (IFN-c) 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-c, 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-c stimulation. The nitric oxide donor S-nitrosoglutathione inhibited antiIgE-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-c 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.

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.

A simplified, nonradioactive procedure for the detection of specific mRNAs on Northern blots has been developed, utilizing digoxigenin-labelled oligonucleotides and chemiluminescence. Antisense oligonucleotide (30-35 mer) probes were designed and synthesised based on published cDNA and gene sequences. These probes were end-labelled (5') with digoxigenin. Total RNA was fractionated by agarose gel electrophoresis and capillary blotted onto positively charged nylon membranes. After hybridization, the mRNA/digoxigenin-labelled oligonucleotide complex was detected by a chemiluminescence-based method using disodium 3-(4-methoxyspiro-[1,2-dioxetane-3-2'(5'chloro)- tricyclo[3.3.1.13.7]decane]-4-yl)phenyl phosphate (CSPD) as substrate. The advantages of this simplified technique for detecting mRNAs in physiological and nutritional studies are described.

IFN-gamma regulates various aspects of rodent peritoneal mast cell function, including mediator release, cell growth, TNF-alpha-mediated cytotoxicity, and MHC class II expression. We investigated whether the suppressive action of IFN-gamma on IgE/Ag-mediated degranulation of mast cells is mediated via synthesis of nitric oxide. Incubation of mouse peritoneal cells with L-NMMA, an inhibitor of nitric oxide synthase, or in medium lacking the nitric oxide precursor L-arginine reversed the inhibitory effect of IFN-gamma on Ag-induced serotonin release. Furthermore, the nitric oxide donors sodium nitroprusside and S-nitrosoglutathione inhibited degranulation, and this effect was direct, since it was seen equally on purified and unfractionated mast cells and occurred independently of IFN-gammaR expression. Additional experiments revealed that accessory cells in peritoneal cell populations were the principal target for the action of IFN-gamma and the main source of nitric oxide; the cytokine was more potent on unfractionated compared with purified mast cells, and IFN-gamma induced detectable nitrite production in mixed peritoneal cells, but not in purified mast cells. These studies show that IFN-gamma induces nitric oxide production in peritoneal cell populations, and that synthesized nitric oxide directly inhibits the IgE-mediated secretory function of mast cells. The activation of nitric oxide-producing cells in the tissue microenvironment may be important in the control of mast cell-dependent allergic reactions.

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