Pharmacologic modulation of inflammatory mediator release by rat mast cells.

Current knowledge of the mechanism of inflammatory mediator release from mast cells is reviewed with particular reference to the role of cyclic nucleotides and calcium and their interrelationship with one another as defined by studies in highly purified rat peritoneal mast cells. Data are presented indicating an important role for intracellular cAMP and calcium in the mediation or modulation of release, as well as evidence for a close relationship between these two regulatory systems. Releasing agents which clearly act at the level of the plasma membrane (concanavalin A and anti-IgE antibody) are shown to differ from releasing agents that may not (48/80 and the ionophore A23187) in regard to the early cellular cAMP response, dependency of the release reaction on phosphatidyl serine, and kinetics of release. Pharmacologic modulators of release are discussed; these include: PGE1 and theophylline, which raise cAMP and inhibit release; and diazoxide, adenine, and carbachol which lower cAMP and potentiate release. Adenosine was also found to enhance release with marked effects at concentrations in the low nanomolar range.     

Mercury induces inflammatory mediator release from human mast cells

Background  

Mercury is known to be neurotoxic, but its effects on the immune system are less well known. Mast cells are involved in allergic reactions, but also in innate and acquired immunity, as well as in inflammation. Many patients with Autism Spectrum Disorders (ASD) have "allergic" symptoms; moreover, the prevalence of ASD in patients with mastocytosis, characterized by numerous hyperactive mast cells in most tissues, is 10-fold higher than the general population suggesting mast cell involvement. We, therefore, investigated the effect of mercuric chloride (HgCl₂) on human mast cell activation.     

Inflammatory cells and mediator release during ragweed challenge: correlation between histamine content in nasal secretions and appearance of inflammatory cells.

The early and late phase responses in the nasal tissues exhibit release of inflammatory mediators and a mixed cellular influx in separate nasal challenges. To explore this phenomenon further, histamine concentration was determined along with characterization of cell influx during dose-dependent ragweed challenges. Ten subjects with allergic rhinitis underwent two unilateral nasal lavages using incremental 3-fold concentrations of short ragweed. Low doses of ragweed (0.016 to 0.114 units Amb a I) rarely induced cell influx (1/18 challenges), whereas moderate doses (0.432 to 1.3 units Amb a I) caused cell influxes in 7/18 and high doses (3.39 to 11.7 units Amb a I) resulted in cell influxes in 8/17. The eluent contained greater than 50% neutrophils in seven challenges; greater than 50% eosinophils in three; and a mixed pattern in six. There was a significant association between the dose of antigen and the level of histamine. Challenges with an eosinophilic influx tended to be associated with higher concentrations of histamine than neutrophilic influxes. Similar to the immediate skin response, the early allergic response in the nose demonstrated a cell influx with release of histamine. Nasal cellular inflammation therefore can occur within minutes of allergen exposure.     

Effects of antigen challenge on intestinal permeability and morphology in rats immunized with gliadin or ovalbumin

In this study we have investigated the possible effects of a local immune response on the intestinal permeability and morphology in rats. The animals were immunized subcutaneously by small doses of gliadin or ovalbumin. Immunization with gliadin led to small but significantly increased levels of IgA and IgM antibodies, similar to those reported for patients with coeliac disease. Immunization with ovalbumin gave significantly increased antibody levels of IgE, IgG, IgA and IgM. Subsequent antigen provocations by oral administration of gliadin (1 mg), together with fluorescein isothiocyanate-labelled dextran (molecular weight 3,000 daltons) as permeability marker, resulted in an increased intestinal permeability for this marker. This alteration of the intestinal permeability was qualitatively similar to that previously reported for patients with coeliac disease. Antigen provocation with ovalbumin caused a decreased permeability for both dextran and different-sized polyethylene glycols, but first at a dose of 40 mg. Direct antigen administration (gliadin or ovalbumin) into a ligated part of the ileum gave qualitatively similar but less pronounced permeability changes when compared with the effects obtained after oral administration of the respective antigen. However, neither gliadin nor ovalbumin challenge led to any ultrastructural changes of the intestinal wall. In summary, we have been successful in inducing specific antibody responses towards gliadin in rats and increased intestinal permeability upon gliadin provocation without any coexisting morphological alterations.     

Ketotifen reduces sneezing but not histamine release following nasal challenge with antigen

We evaluated the effect of pre-treatment with 1 and 2 mg b.i.d. of ketotifen on the early response to nasal challenge in a double-blind cross-over trial. Weekly nasal challenges were performed in 10 allergic subjects after 1 hr and 1, 2, 3 and 4 weeks of ketotifen administration. The response to nasal challenge was monitored by counting the number of sneezes, the assessment of subjective symptoms, and by measuring the levels of histamine and TAME-esterase activity in recovered nasal lavages. The number of sneezes diminished significantly after a single dose of drug with both the 1 and 2 mg doses. Prolonged pre-treatment did not improve the results. The levels of histamine and TAME-esterase activity in recovered nasal lavages were not changed significantly by either pre-treatment at either dose. Although the number of subjects was small, our data suggest that ketotifen diminishes allergic symptomatology by its antihistaminic properties rather than by inhibiting histamine release from mast cells. As we did not look into the effects of ketotifen on other products generated by mast cells (prostanoids, leukotrienes and tryptase), we cannot fully rule out an effect on mast cells.     

Effect of antigen dose on the recruitment of inflammatory cells to the lung by segmental antigen challenge.

Bronchoscopic antigen challenge of atopic volunteers results in an immediate release of inflammatory mediators and, after a number of hours, the recruitment of inflammatory cells to the lung. The purpose of this work was to investigate the effect of antigen dose on the subsequent recruitment of inflammatory cells to the lung. Twenty-two volunteers without asthma, eight nonatopic control subjects, and 14 ragweed-allergic subjects underwent 25 local antigen-challenge procedures that consisted of a baseline lavage of a control segment, antigen challenge of another segment in the contralateral lung, and lavage of the challenged segment 24 hours later. A 25,000-fold range of antigen doses was used from 0.004 to 100 PNU/ml (0.02 to 500 ng/ml of ragweed antigen E [Amb a I]). Challenge of nonatopic control subjects resulted in the recruitment of only a small number of inflammatory cells, less than a twofold increase in comparison with the cells of control lavage; this increase was primarily due to an increase in neutrophils. Challenge of atopic subjects, in contrast, resulted in approximately a threefold to ninefold increase in inflammatory cells with more cells recruited at larger doses of antigen. Only subjects challenged with a "high" dose of antigen (greater than or equal to 1 PNU/ml) recruited significant quantities of eosinophils to the lung. In these subjects, a twofold increase in macrophages, a fourfold increase in lymphocytes, a 90-fold increase in neutrophils, and an 800-fold increase in eosinophils were observed; the number of neutrophils and eosinophils recruited averaged between 30 and 60 million.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of protein phosphorylation and mediator secretion following antigen challenge of mast cells sensitized with monoclonal IgE or with reaginic serum

Inflammation Research -     

The immunogenic capacity of antigen taken up by peritoneal exudate cells

The immunogenic capacity of protein antigens has been compared for the free and peritoneal exudate cell (PEC)-bound forms. The response of CBA mice to BSA provides the reference system, but lysozyme, ovalbumin, HSA and modified BSA were also studied. Uptake is approximately equally efficient in vivo and in vitro. PEC-bound antigen, estimated by radioactivity is far more potent than the free form in inducing primary immunization. The following properties were also found: (i) viable PEC are required; (ii) irradiation of the cell donor 2–7 days before giving antigen inhibits immunization, but irradiation after uptake does not do so; (iii) mice are susceptible to immunization during their phase of recovery from paralysis; (iv) PEC do not retain large amounts of antigen for long; (v) the activity does not depend solely on a minor, phagocytosisprone fraction of the antigen; (vi) allogeneic transfer of PEC reduces their immunogenic capacity; (vii) paralysed hosts are not susceptible to immunization, but PEC from paralysed donors are effective; and (viii) the enhancement of immunogenic capacity does not apply to the secondary response. The conclusion may be drawn that the retention of small quantities of antigen by macrophages plays an essential role in some, but probably not all types of immune response.     

Pore formation by the Escherichia coli alpha-hemolysin: role for mediator release from human inflammatory cells.

The Escherichia coli alpha-hemolysin represents a potent stimulus for inflammatory mediator release (O2-, beta-glucuronidase release, and leukotriene generation) from human polymorphonuclear granulocytes, for histamine release from a suspension of human lymphocyte/monocyte basophil cells (LMB), and for serotonin release and 12-hydroxyeicosatetraenoic acid generation from human platelets. In contrast, the E. coli alpha-hemolysin leads to a downregulation of cytokine release (interleukin-1 beta [IL-1 beta], IL-6, and tumor necrosis factor alpha) from human LMB. Recently, it became apparent that the E. coli alpha-hemolysin is composed of several functional structures. We analyzed the role of pore formation, pore stability, and calcium-dependent membrane binding for inflammatory mediator release by using washed bacteria as well as culture supernatants of isogenic recombinant E. coli strains expressing no hemolysin (Hly-), the wild-type hemolysin (Hly+), or hemolysin molecules deficient or modulated in defined functions (pore formation, calcium-dependent membrane binding, or pore stability). In human granulocytes and platelets, mutant hemolysin with enhanced pore stability did not lead to a further increase in induction; mutant hemolysin deficient in pore-forming activity or calcium-dependent membrane binding no longer induced leukotriene B4 generation or beta-glucuronidase release compared with the wild-type hemolysin. Similar results were obtained with regard to histamine release from human LMB. The induction of cytokine release from human LMB differed depending on the type of mutant E. coli alpha-hemolysin. The wild-type hemolysin, the mutant hemolysin with enhanced pore-forming activity, and, to a lesser degree, the mutant hemolysin deficient in pore-forming activity decreased cytokine release (IL-1 beta, IL-6, IL-8, and tumor necrosis factor) compared with untreated cells. In contrast, the mutant hemolysin deficient in calcium-dependent membrane binding led to an increase of up to 50% in cytokine release compared with that by unstimulated cells. Our results indicate that simultaneous expression of the pore-forming and calcium-dependent membrane-binding activities of the hemolysin molecule was necessary to obtain the full cellular inflammatory response pattern observed with the wild-type hemolysin.     

Tumor immunity in rats immunized with rat carcinoembryonic antigen

Active immunization of rats with an emulsion consisting of Freund's complete adjuvant (FCA) and an extract of rat tumor containing carcinoembryonic antigen (CEA) induced clear-cut protection from growth of the syngeneic CEA-positive tumor, RCA-1. No protection was observed in rats treated with FCA alone nor was there protection against a tumor that no serologically detectable CEA. The results suggested that the tumor immunity exhibited by the immunized rats was mediated by an immune response specific for rat CEA. It was shown further that multiparous rats were more resistant to growth of RCA-1 tumor than nulliparous rats. This suggested that immunization against rat CEA, which is an oncofetal antigen, may occur during pregnancy.     

Mast cell mediator release in nonasthmatic subjects after endobronchial adenosine challenge

BACKGROUND: Adenosine 5'-monophosphate (AMP) has been shown to cause bronchoconstriction in atopic subjects but to have no effect on nonatopic nonasthmatic subjects. Endobronchial AMP challenge has previously been shown to cause mast cell mediator release in asthmatic subjects, but it is unknown whether a similar response occurs in atopic nonasthmatic and nonatopic nonasthmatic control subjects who have no response to inhalation AMP challenge. OBJECTIVE: This study examined the change in mast cell-derived products after endobronchial saline challenge and AMP challenge in subjects with and without a positive inhalation response to AMP. METHODS: Inhalation challenge with AMP challenge was performed in normal, atopic nonasthmatic, and atopic asthmatic subjects. Levels of mast cell mediators were measured after endobronchial adenosine challenge and after placebo endobronchial saline challenge. RESULTS: There were significant increases in histamine, tryptase, protein, and prostaglandin D2 levels (P=.02, P=.02, P=.01, and P=.01, respectively) after AMP challenge compared with after saline challenge in nonatopic nonasthmatic subjects. There was no significant increase in any mediator in either of the other 2 groups. CONCLUSION: This study suggests dissociation between mediator release and bronchoconstriction in response to AMP.     

Effects of the prostaglandin analogue misoprostol on inflammatory mediator release by human monocytes

The effect of misoprostol (M) on IL-1, TNF-, and lipid mediator release (assessed by RIA) by adherent (assessed by electron microscopy) human monocytes were studiedin vitro. Human monocytes stimulated with E. Coli-derived lipopolysaccharide showed an increase in both IL-1 and TNF- release. Incubation of the monocytes with LPS and M (18 hrs.), resulted in a reduction of both IL-1 and TNF- levels. Leukotriene B₄ levels did not increase in response to LPS or M. LPS also caused an increase in thromboxane (TXB₂).M decreased TXB₂ levels. 6-keto PGF1 (6KP). Incubation with LPS and M stimulated release. LPS caused an increase in PGE₂ levels. M (100 M) caused an increase in PGE₂ levels, M (1 M) had no effect on PGE₂. These data suggest a possible immunomodulatory role for misoprostol in inflammatory diseases.     

Antigen-induced local mediator release and cellular inflammatory responses in atopic subjects

We report comparisons of histamine release and neutrophil exudation in skin-window sites of 27 pollen-sensitive subjects challenged with antigen or buffer. More histamine was released within 30 min into appended collection chambers in antigen sites vs control sites. There were also more neutrophils adhering to membrane filters applied for 1 hr to the blister bases in antigen-challenged sites vs buffer sites. Comparison of skin-test extinction dilution titer, histamine release, and neutrophil accumulation in antigen-challenged sites in individual subjects showed that (1) there was no correlation between degrees of local histamine and neutrophil accumulation, (2) the increase in histamine but not in neutrophils correlated inversely with the concentration of antigen required to elicit a minimum wheal, and (3) both histamine and neutrophil increases were induced by antigen in a dose-dependent manner.     

siRNA沉默MIF基因对糖皮质激素抑制脂质炎症介质释放的影响及其机制

 目的： 研究小干扰RNA（siRNA）阻断巨噬细胞移动抑制因子（macrophage migration-inhibitory factor,MIF）基因表达对糖皮质激素抑制脂质炎症介质释放的影响及其细胞内机制。方法：体外培养小鼠巨噬细胞系RAW2647,采用免疫荧光法观测siRNA转染效率,RT-PCR检测MIF mRNA的表达,Western blotting检测MIF蛋白的表达;RAW2647细胞转染MIF siRNA后观察地塞米松（Dex）抗炎作用的变化,用ELISA检测细胞上清中前列腺素E2(PGE2)和白三烯B4(LTB4)的含量,Western blotting检测胞浆膜联蛋白Annexin 1和下游胞浆磷酸酯酶A2α（cPLA2α）的蛋白表达变化。结果：与阴性对照相比,MIF siRNA能有效阻断细胞内源性MIF蛋白的表达,增强RAW2647细胞对Dex作用的敏感性;明显增强Dex抑制PGE2和LTB4产生的效应,增加胞浆蛋白Annexin 1的表达,抑制cPLA2α的磷酸化。结论：MIF siRNA能增强糖皮质激素抑制脂质炎症介质PGE2和LTB4的释放,且可能是通过影响Annexin 1-cPLA2α信号通路实现的。阻断内源性MIF蛋白的表达可显著增强RAW2647细胞对糖皮质激素抗炎作用的敏感性。