In the present work, we have examined the effect of PAF, removal of epithelium, the mechanism of desensitization, and the substances that increases the level of intracellular c-AMP on the differences of mediator release from superfused tracheal strips after passive sensitization with IgG1 versus IgE Ab. In the passive sensitized tracheal tissues, the effect of PAF and the mechanism of desensitization have been examined by PAF antagonist, CV 3988 and DFP, respectively. The epithelium was stripped from one-half of each trachea by mechanical means. Both superfused tracheal tissues were challenged with Ox-Ag. Inhibitors of mediator release were added into a superfused buffer. Hist released was determined by spectrophotofluorometer, and LT by radioimmunoassay. PAF known to mediate the allergic reaction was not released by Ag after both Ab sensitization. Epithelium removal resulted in similar contraction, Hist and LT release after IgG1 Ab activation, but in the IgE Ab activation, epithelium removal resulted in smaller contraction and Hist release. In the L-cysteine and indomethacin pretreatment after two Ab sensitization, epithelium removal decreased the release of Hist and LT. The compound 48/80 pre-challenge and epithelium removal resulted in the increase of Hist release, but in the decrease of LT release after IgG1 or IgE sensitization. The Amount of LT released by Ag after compound 48/80 pre-challenge increased in the absence or presence of epithelium after both Ab sensitization. Mediator release from tissues sensitized with both Abs was not changed by DFP. The responses of inhibitors to prevent the mediator release were more effective on the IgE Ab than on the IgG1 Ab sensitization. These studies suggest that the tracheal epithelium can act to inhibit immune- and non-immune-induced airway responses. Non-immunological responses may in part reflect the role of epithelium as a diffusion barrier and modulator of mediator release. These data also suggest that immunological responses are related to the localization and functional heterogeneity of tissue mast cells.
Animal ; Epithelium/immunology/physiology ; Female ; Guinea Pigs ; *Histamine Release ; *Immunization ; Immunoglobulin E/*immunology ; Immunoglobulin G/*immunology ; Leukotrienes/metabolism ; Mast Cells/immunology ; Support, Non-U.S. Gov't ; Trachea/*immunology/physiology

BACKGROUND: It has been reported that Korean Red Ginseng saponins are effective in increasing the synthesis of serum proteins, in cellular proliferation, in producing antibody against sheep red blood cells, and in various cancer. However, there have been no reports yet on the immunomodulating activity of this allergic disease. OBJECTIVE: This study was conducted to find the immunomodulating activity of a single highly purified ginsenoside, Rb1, by using ovalbumin(OVA) as the antigen. METHOD: BALB/c mice were immunized subcutaneously with OVA containing the alum or ginsenoside, Rb1, twice per 2wk interval. Antigen-specific antibodies and IgG subclasses were determined from serum recovered by cardiac puncture 2 wk after the second immunization by using ELISA method. Antigen-specific cellular proliferation and cytokines were quantified from splenotes obtained from spleens immunized. Cytokine level in cell culture supernatants were determined by ELISA method. NK cell cytotoxicity was generated by co-culture of splenic mononuclear cells against YAC-1 cells as target cells. Hemolytic activity of Rb1 was determined by an in vitro assay using sheep red blood cells. RESULTS: BALB/c mice immunized with OVA plus Rb1 produced significantly higher titers of antibodies than mice immunized with alum-adsorbed antigen. Rb1 remarkably increased titers in IgG2a and IgG2b subclasses. Antigen-specific proliferative response was more significantly increased with the use of Rb1 than with alum-adsorbed antigen. Rb1 reduced IL-4 production, increased IL-10 production more than alum-adsorbed OVA, but did not affect the IFN-gamma production. High concentration of Rb1 increased the splenic mononuclear cells that were capable of killing YAC-1 cells. Rb1 did not stimulate the production of reaginic antibody (IgE) but alum was able to induce it. Rb1 did not show any hemolytic activity up to 500microgram/ml. CONCLUSION: The data suggest that the highly purified ginsenoside extracted from Korean Red Ginseng Radix, Rb1, can actively influence the switch of Igs produced by OVA. The data also suggest that Rb1 may be an immunosurveillant in NK cytotoxic activity.
Animals ; Antibodies ; Blood Proteins ; Cell Culture Techniques ; Cell Proliferation ; Coculture Techniques ; Cytokines ; Enzyme-Linked Immunosorbent Assay ; Erythrocytes ; Homicide ; Immunization ; Immunoglobulin G ; Interleukin-10 ; Interleukin-4 ; Killer Cells, Natural ; Mice ; Ovalbumin ; Ovum ; Panax ; Punctures ; Saponins ; Sheep ; Spleen

The inhibitory effect of cromakalim on the mediator release from mast cells caused by antigenantibody reactions was in controversy with the specific antigen used. However, it has recently been observed that cromakalim inhibits the release of mediators from superfused tracheal and parenchymal strips or lung mast cells after passive sensitization with the IgG1 antibody. An attempt, therefore, was made to determine the inhibitory mechanisms of cromakalim on the release of mediators such as histamine and leukotriene released by the activation of enzymes during mast cell activation. Guinea pig lung mast cells were purified through enzyme digestion, rough percoll and continuous percoll density gradients. The purified mast cells were prelabeled with [3H]palmitic acid. PLD activity was assessed more directly by the production of labeled phosphatidylethanol by PLD-mediated transphosphatidylation in the presence of ethanol. In the cells labelled with [3H]myristic acid, [3H] DAG production was measured. The methyltransferase activity was assessed by measuring the incorporation of [3H]methyl moiety into phospholipids in sensitized mast cells labelled with L-[3H] methylmethionine. cAMP level was measured by radioimmunoassay. Cromakalim resulted in a decrease in the amount of histamine and leukotrienes releases by 30% in the ovalumin-induced mast cell. Cromakalim had little effect on phospholipase D activity enhanced by the activated mast cell. Cromakalim inhibited the initial increase of diacylglycerol production during mast cell activations. Cromakalim inhibited the phospholipid methylation increased in the activated mast cell. These results show that cromakalim decreases histamine release by inhibiting the initial increase of 1,2-diacylglycerol during the mast cell activation, which is mediated via the phosphatidylinositide-phospholipase C system rather than the phosphatidylcholine-phospholipase D system. Furthermore, cromakalim reduces phosphatidylcholine production by inhibiting the methyltransferase, which decreases the conversion of phosphatidylcholine into arachidonic acid and inhibits the production of leukotrienes.
Adenylate Cyclase/metabolism ; Animal ; *Antigen-Antibody Reactions ; Benzopyrans/*pharmacology ; Cromakalim ; Diglycerides/biosynthesis ; Female ; Guinea Pigs ; Histamine Release/*drug effects ; Leukotrienes/*secretion ; Lung/drug effects/secretion ; Mast Cells/*drug effects/secretion ; Methylation ; Phospholipase D/metabolism ; Phospholipids/metabolism ; Potassium Channels/*drug effects ; Pyrroles/*pharmacology ; Support, Non-U.S. Gov't

The inhibitory effect of cromakalim on the mediator release from mast cells caused by antigenantibody reactions was in controversy with the specific antigen used. However, it has recently been observed that cromakalim inhibits the release of mediators from superfused tracheal and parenchymal strips or lung mast cells after passive sensitization with the IgG1 antibody. An attempt, therefore, was made to determine the inhibitory mechanisms of cromakalim on the release of mediators such as histamine and leukotriene released by the activation of enzymes during mast cell activation. Guinea pig lung mast cells were purified through enzyme digestion, rough percoll and continuous percoll density gradients. The purified mast cells were prelabeled with [3H]palmitic acid. PLD activity was assessed more directly by the production of labeled phosphatidylethanol by PLD-mediated transphosphatidylation in the presence of ethanol. In the cells labelled with [3H]myristic acid, [3H] DAG production was measured. The methyltransferase activity was assessed by measuring the incorporation of [3H]methyl moiety into phospholipids in sensitized mast cells labelled with L-[3H] methylmethionine. cAMP level was measured by radioimmunoassay. Cromakalim resulted in a decrease in the amount of histamine and leukotrienes releases by 30% in the ovalumin-induced mast cell. Cromakalim had little effect on phospholipase D activity enhanced by the activated mast cell. Cromakalim inhibited the initial increase of diacylglycerol production during mast cell activations. Cromakalim inhibited the phospholipid methylation increased in the activated mast cell. These results show that cromakalim decreases histamine release by inhibiting the initial increase of 1,2-diacylglycerol during the mast cell activation, which is mediated via the phosphatidylinositide-phospholipase C system rather than the phosphatidylcholine-phospholipase D system. Furthermore, cromakalim reduces phosphatidylcholine production by inhibiting the methyltransferase, which decreases the conversion of phosphatidylcholine into arachidonic acid and inhibits the production of leukotrienes.
Adenylate Cyclase/metabolism ; Animal ; *Antigen-Antibody Reactions ; Benzopyrans/*pharmacology ; Cromakalim ; Diglycerides/biosynthesis ; Female ; Guinea Pigs ; Histamine Release/*drug effects ; Leukotrienes/*secretion ; Lung/drug effects/secretion ; Mast Cells/*drug effects/secretion ; Methylation ; Phospholipase D/metabolism ; Phospholipids/metabolism ; Potassium Channels/*drug effects ; Pyrroles/*pharmacology ; Support, Non-U.S. Gov't

The pathogenesis of chronic idiopathic urticaria is not completely understood, but mast cell degranulation and histamine release are thought to be of central importance. It is now established that circulating autoantibodies against the high-affinity IgE receptor(Fc(epsilon)RIalpha) can be found approximately one third of patients with chronic idiopathic urticaria. These autoantibodies can be detected by in vivo autologous serum skin test and by in vitro basophil and mast cell histamine release assays as functional tests, and also can be confirmed by in vitro enzyme-linked immunosorbent assay to Fc(epsilon)RIalpha and Western blot analysis. Our purpose was to determine the proportion of patients with positive autologous serum skin test and anti-Fc(epsilon)RIalpha antibody in chronic idiopathic urticaria and whether there are differences between patients with and those without autoantibodies in the clinical features. RESULTS ARE AS FOLLOWS: 1. Positive result to autologous serum skin test was 58.5% in 41 patients of chronic idiopathic urticaria. There was no significant difference of clinical features and laboratory tests between patients with positive skin test and those with negative results. 2. By enzyme-linked immunosorbent assay, anti-Fc(epsilon)RIalpha antibody was detected in sera from 34% of patients with chronic idiopathic urticaria. 3. In sera from 33% of patients with positive skin test and 35% of those with negative result, we could demonstrate anti-Fc(epsilon)RIalpha antibody by enzyme-linked immunosorbent assay. 4. There was no differences of clinical features and laboratory tests between the patients with autoantibodies to Fc(epsilon)RIalpha and those without, except female predominance and longer urticaria history in those with autoantibodies.
Autoantibodies ; Basophils ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Female ; Histamine Release ; Humans ; Immunoglobulin E* ; Mast Cells ; Skin Tests ; Urticaria*

Heat contact urticaria is very rare and it is characterized by the development of wheal limited to the areas of heat contact. We report a case of heat contact urticaria in a 65-year-old women. The wheal was induced by hot bathing, washing in hot water or leaning on hot radiators. Symptoms started within 5 minutes of exposure and lasted 30 to 60 minutes. She had no systemic symptoms. The clinical diagnosis of localized heat urticaria was confirmed by experimental induction of localized wheals. Our investigation showed that the threshold temperature needed for induction of the heat urticaria was 39 degrees C. We tried to investigate the plasma levels of prostaglandin D2 and blood histamine before and after heat challenge. The patient showed marked improvement after a combination treatment of desensitizing by repeated exposure to heat and indomethacine.
Aged ; Case Report ; Female ; Heat/*adverse effects ; Human ; Prostaglandin D2/blood ; Urticaria/*etiology/therapy

For those with allergy, vaccination with a specific allergen has often been used as a major therapeutic measure. However, the universal application of this technique in clinics have been restricted due to its low success rates and the risk of active systemic anaphylactic shock (ASAS). In this regard, we constructed a fusion protein (OVA-DT), ovalbumin (OVA) fused with diphtheria toxin protein (DT), which may exert a specific cytotoxicity to cells bearing OVA-specific IgE. Its therapeutic effect was evaluated in mice (BALB/c) sensitized with OVA (Os-mice). OVA challenges to the OVA-sensitized mice (Os-mice) caused ASAS to death within 30 min, but OVA-DT treatment afforded mice complete protection. When OVA-DT was treated to the Os-mice, none showed the signs of ASAS when re-challenged 48 h after the treatment. OVA-DT itself was not found to be toxic or allergenic in normal mice. The effect of OVA-DT on the biological functions of mast cells was also studied. Binding of OVA-DT to OVA-specific IgE bearing mast cells and the inhibition of histamine release from these cells were observed. In addition, OVA-DT treatment inhibited the proliferation of OVA-specific B cells in mice. In Os-mice treated with OVA-DT, levels of anti-OVA IgG2a in serum and the production of IFN-gamma by splenic lymphocytes were found to increase, but the production of IL-4 by these cells decreased. Re-direction of cytokine profiles from OVA-specific Th2 to OVA-specific Thl is suggested. These results indicate that OVA-DT can protect Os-mice from ASAS due to OVA challenge, because it inactivates OVA-specific IgE-expressing cells, including mast cells and B cells.
Anaphylaxis/prevention | control* ; Animal ; B-Lymphocytes/immunology ; Female ; Histamine Release/drug effects ; IgE/metabolism ; Interferon Type II/biosynthesis ; Interleukin-4/biosynthesis ; Lymphocyte Transformation/drug effects ; Mast Cells/metabolism ; Mice ; Mice, Inbred BALB C ; Ovalbumin/immunology* ; Recombinant Fusion Proteins/therapeutic use*

We previously reported that some components of ginsenosides decreased mediator releases evoked by the activation of mast cells with specific antigen-antibody reactions. This study aimed to assess the effects of ginsenosides (Rb2, Re) on the mechanism of histamine release in the mast cell activation. We partially purified guinea pig lung mast cells by using enzyme digestion, the rough and the discontinuous percoll density gradient method. Mast cells were sensitized with IgG1 and challenged with ovalbumin (OA). Histamine was assayed by fluorometric analyzer, leukotrienes by radioimmunoassay. Phospholipase D (PLD) activity was assessed more directly by the production of (3H)phosphatidylbutanol (PBut) which was produced by PLD-mediated transphosphatidylation in the presence of butanol. The amount of 1,2-diacylglycerol (DAG) were measured by the (3H)DAG labeled with (3H)palmitic acid or (3H)myristic acid. Pretreatment of Rb2 (300 microgram) significantly decreased histamine release by 60%, but Re (300 gg) increased histamine release by 34%. Leukotrienes release in Rb2 was decreased by 40%, Re was not affected in the leukotrienes release during mast cell activations. An increasing PLD activity during mast cell activation was decreased by the dose-dependent manner in the pretreatment of Rb2, but Re pretreatment facilitated the increased PLD activity during mast cell activation. The amount of DAG produced by phospholipase C (PLC) activity was decreased by Rb2 pretreatment, but Re pretreatment was not affected. The amount of mass DAG was decreased by Rb2 and Re pretreatment during mast cell activation. The data suggest that Rb2 purified from Korean Red Ginseng Radix inhibits the DAG which is produced by the activation of mast cells with antigen-antibody reactions via both phosphatidylinositide-PLC and phosphatidylcholine-PLD systems, and then followed by the inhibition of histamine release. However, Re increases histamine release by stimulation of DAG production, which is mediated by phosphatidylcholine-PLD system rather than by phosphatidylinositide-PLC system, but inhibits the mass DAG production. Thus, it could be inferred that other mechanisms play a role in the increase of histamine release during mast cell activation.
Animals ; Antigen-Antibody Reactions* ; Digestion ; Ginsenosides* ; Guinea Pigs* ; Guinea* ; Histamine Release* ; Histamine* ; Immunoglobulin G ; Leukotrienes ; Lung* ; Mast Cells* ; Ovalbumin ; Panax ; Phospholipase D ; Radioimmunoassay ; Type C Phospholipases

Cromakalim (BRL 34915), known as an airway smooth muscle relaxant, inhibited the releases of mediators in the antigen-induced mast cell activation. It has been suggested that cromakalim, in part, inhibited mediator releases by inhibiting the initial increase of 1,2-diacylglycerol (DAG) produced by the activation of the other phospholipase system which is different from phosphatidylcholine-phospholipase D pathway. The aim of this study is to further examine the inhibitory mechanism of cromakalim on the mediator release in the mast cell activation. Guinea pig lung mast cells were purified by using enzyme digestion and percoll density gradient. In purified mast cells prelabeled with (3H)PIP2, phospholipase C (PLC) activity was assessed by the production of (3H)insitol phosphates. Protein kinase C (PKC) activity was assessed by measuring the protein phosphorylated from mast cells prelabeled with (gamma-32P)ATP, and Phospholipase A2 (PLA2) activity by measuring the lyso-phosphatidylcholine produced from mast cell prelabeled with 1-palmitoyl-2-arachidonyl phosphatidyl-(14C)choline. Histamine was assayed by fluorometric analyzer, and leukotrienes by radioimmunoassay. The PLC activity was increased by activation of the passively sensitized mast cells. This increased PLC activity was decreased by cromakalim pretreatment. The PKC activity increased by the activation of the passively sensitized mast cells was decreased by calphostin C, staurosporine and cromakalim, respectively. The PLA2 activity was increased in the activated mast cells. The pretreatment of cromakalim did not significantly decrease PLA2 activity. These data show that cromakalim inhibits histamine release by continuously inhibiting signal transduction processes which is mediated via PLC pathway during mast cell activation, but that cromakalim does not affect PLA2 activity related to leukotriene release.
Animals ; Antigen-Antibody Reactions* ; Cromakalim* ; Digestion ; Guinea Pigs* ; Guinea* ; Histamine ; Histamine Release ; Leukotrienes ; Lung* ; Mast Cells* ; Membranes* ; Muscle, Smooth ; Phosphates ; Phospholipases ; Phospholipases A2 ; Protein Kinase C ; Radioimmunoassay ; Signal Transduction ; Staurosporine ; Type C Phospholipases

Chronic spontaneous urticaria (CSU) is a complex idiopathic disease of the skin with various cellular infiltrations. Although mast cells are key effector cells in the pathogenesis of CSU, CD4+ T helper 2 cells also have particular roles in the development and maintenance of CSU. Periostin is known as a downstream molecule of interleukin (IL)-4 and IL-13, key cytokines of type 2 immune responses. In this study, we examined periostin and IL-13 levels in the sera of patients with CSU (n=84) and healthy normal controls (NCs, n=43). Periostin levels were significantly lower in the CSU group than in NCs (71.4±21.8 vs 85.1±22.4 ng/mL, P=0.04). Periostin levels were also lower in the severe CSU group than those in mild CSU (59.7±18.0 vs 73.4±22.0 ng/mL, P=0.04). However, IL-13 levels were significantly higher in patients with CSU than in NCs (508.5±51.2 vs 200.7±13.3 pg/mL, P=0.001). In conclusion, periostin and IL-13 may be independently related to the pathogenesis of CSU.
Cytokines ; Humans ; Interleukin-13* ; Interleukins ; Mast Cells ; Skin ; Urticaria*