Sodium orthovanadate was found to be an effective histamine liberator from serosal mast cells of the rat and mouse. The release process was slow, non-cytotoxic and strongly dependent on pH and extracellular calcium. The effect was highly tissue and species specific and human basophil leucocytes, human lung mast cells and tissue mast cells of the rat and guinea pig were only weakly responsive or essentially unreactive. Other oxyanions of vanadium with the metal in the (+V) oxidation state also evoked histamine release from rat peritoneal mast cells but neither vanadyl sulphate (+IV oxidation state) nor the analogous orthophosphate anion were effective secretagogues. On the basis of these results, the possible mechanism of action of vanadate is discussed.
Human isolated lung preparations were passively sensitized using mouse monoclonal dinitrophenyl (DNP)-specific IgE antibodies. The contractile response to antigen (DNP-bovine serum albumin; DNP-BSA) was approximately 80% of the histamine response (50 microM: 0.20 +/- 0.03 g/mm2) in bronchial muscle preparations. In passively sensitized pulmonary vascular and parenchymal preparations, the contraction to antigen was negligible. Indomethacin (1.7 microM; 30 min) did not alter the contractile response to DNP-BSA (5 micrograms/ml) in bronchial tissues. In passively sensitized bronchial muscle preparations stimulated with DNP-BSA, there was a significant increase (2-fold) in prostanoid production. This production was inhibited by indomethacin. These data suggest that endogenous prostaglandins may not play a role in the regulation of human isolated bronchial muscle contraction to antigen in vitro.
Endogenous and exogenous free radical scavengers significantly decrease mast cell histamine release induced by coincubation with resting, activated platelets or with platelet-derived supernatant. Histamine and pyridylethylamine dose-dependently enhance platelet aggregation; the effect is potentiated by ranitidine and blocked by mepyramine. Histamine increases also cytosolic calcium concentration in platelets stimulated with thrombin, and binding sites for [3H]-mepyramine are present on platelet membranes.
Phytohemagglutinin (PHA)-induced human T cell clones (TCC) derived from conjunctival flogistic tissues of 3 patients with vernal conjunctivitis produced unusually high amounts of interleukin-4 (IL-4) and no, or limited amounts of, gamma-interferon (IFN-gamma). Allergen (Dermatophagoides pteronyssinus or Lolium perenne group I)-specific TCC derived from peripheral blood of two atopic donors produced significantly higher amounts of IL-4 and significantly lower amounts of IFN-gamma than TCC specific for bacterial antigens (tetanus toxoid and PPD) contemporarily established from the same donors. These data provide evidence for a compartimentalization of Th2-like helper T cells in target organs and in the allergen-specific T cell repertoire of allergic patients. Non-B, non-T bone marrow cells could produce IL-4, but not IL-2 or IFN-gamma, in response to cross-linkage of Fc epsilon type I receptors. These cells may further contribute to the maintenance and amplification of allergic inflammation.
Cross-allergenicity between five cereal grains including rice, wheat, corn, Japanese millet (Panicum crus-galli L. var. frumentaceum Trin.) and Italian millet (Setaria italica Beauv. var. germanica schrad.) was examined by radioallergosorbent test (RAST) and RAST inhibition assay. There were significant close correlations between every combinations of RAST values for the five cereal grain extracts. RAST inhibition assay of each extract against RAST discs coupled with other cereal grain extracts indicated marked cross-reactivity of IgE binding between these cereal grain extracts. Rice protein 16KD (RP16KD) was shown to be one of major allergens in rice grain extracts by immunoblotting analysis, histamine release assay from human leukocytes and RAST inhibition. Next, the involvement of RP16KD in the cross-allergenicity between these cereals was investigated. RAST values for RP16KD significantly correlated with that for Italian millet as well as rice but not with those for corn and wheat. There was a trend of positive correlation between RAST values for RP16KD and Japanese millet. In the RAST inhibition assay using sera with positive RAST for these five cereal grain extracts and RP16KD, RP16KD inhibited IgE binding to these all cereal discs in a dose-dependent manner. Similarly, all of the five cereal grain extracts showed an effective decrease in IgE binding to the RP16KD disc. These results indicated possible participation of IgE binding structure on RP16KD in cross-allergenicity between these cereal grain extracts in the Poaceae family.
Abnormalities of peripheral-blood lymphocyte subsets and activation markers were detected in patients with both allergic and nonallergic asthma. Most allergic asthmatics were characterized by increased numbers of IL-2R+ helper T cells and CD23+ B cells. In contrast, nonallergic asthmatics showed increased numbers of IL-2R+ and HLA-DR+ helper and cytotoxic T cells, and a clear redistribution from naive (CD45RA+) to memory (CD45RO+) cells. The number of IL-2R+ T cells correlated with the number of CD23+ B cells in allergic asthma. These changes in the distribution and activation state of T cells suggest an active role for T cells in the pathogenesis of both allergic and nonallergic asthma.
Mast cells are involved in allergic reactions where they release numerous vasoactive and other mediators in response to IgE and antigen. They are also activated by neuropeptides and are found in close contact with neurons. Mast cell heterogeneity has now been documented for mucosal mast cells and connective tissue mast cells. Rat brain mast cells were studied in a perfusion system and were shown to release serotonin in response to the mast cell secretagogue compound 48/80 (C48/80). High-potassium neuronal depolarization also released serotonin, but this was calcium dependent, not associated with beta-hexosaminidase, and was unaffected by prior treatment with C48/80. Neuronal depolarization, however, was associated with somatostatin secretion and substantially reduced subsequent C48/80 stimulation, an effect abolished by neonatal treatment of the animals with capsaicin. Perfusion with somatostatin and substance P also induced brain mast cell serotonin release. C48/80 stimulation of combined thalamic and hypothalamic slices after neuronal depolarization substantially reduced the C48/80 effect, suggesting the possible presence of endogenous inhibitors released from the hypothalamus. Finally, the alpha-2-receptor agonist clonidine had a slight stimulatory effect. These results indicate that brain mast cell serotonin release may be regulated by endogenous neurotransmitters and/or neuromodulators.
Inflammatory cells like eosinophils, neutrophils or mononuclear phagocytes have long been recognized as essential components in the pathophysiology of asthma. After recruitment in situ and subsequent activation, they are considered as responsible for epithelial and submucosal bronchial alterations. However, to access to the inflammatory site, these cells have to cross the endothelial wall, suggesting so a potential implication of endothelial cells (EC) in bronchial asthma. To test this hypothesis, we studied in a first step the modulation of vascular adhesions like intercellular adhesion molecule-1 (ICAM-1) on EC: supernatants of alveolar macrophages (AM) recovered by bronchoalveolar lavage in patients exhibiting a late asthmatic reaction, induced an enhanced expression of ICAM-1 on EC preparations; increase of ICAM-1 was clearly correlated to amounts of tumor necrosis factor-alpha (TNF-alpha) present in AM supernatants, as shown by inhibition experiments with anti-TNF-alpha antiserum. The second way to explore the possible role of EC in asthma was the detection of autoantibodies to EC in various allergic disorders: antibodies against a 120-kD EC antigen in patients with allergic granulomatosis and angiitis, antibodies towards a 55-kD component, common to human EC and platelets in patients with severe asthma, namely characterized by their corticosteroid dependence or by aspirin-induced intolerance. So our data suggest that bronchial asthma might result from either EC activation, through the induction of surface adhesion molecules or from an autoimmune process involving EC antigens.
IgG autoanti-IgE is detectable in a large proportion of individuals with allergic asthma, where it is suggested to be potentially involved in modulating IgE-mediated hypersensitivity. Using a series of overlapping recombinant human epsilon-chain peptides, we have shown that circulating IgG anti-IgE antibodies recognise at least 2 epitopes located within the C epsilon 2 and the C epsilon 4 domains, respectively. The C epsilon 2 recognition site is located within the C-terminal portion of the C epsilon 2 domain (i.e. aa301-339) which is thought to contribute residues to the Fc epsilon RI-binding site on IgE. The recognition by autoanti-IgE of an effector function site of such pivotal importance in IgE-mediated hypersensitivity suggests that it plays a possible modulatory role during mast cell and basophil activation.
In pulmonary sarcoidosis an activation of alveolar T lymphocytes and alveolar macrophages (AM) has been demonstrated. There is evidence that in contrast to acute disease a heightened T-cell response cannot be observed in the chronic phase of sarcoidosis. The role of AM in the inflammatory process of chronic sarcoidosis is not yet intensively evaluated. To address this question we measured the release of tumor necrosis factor alpha (TNF alpha) and interleukin-1 (IL-1) by AM of 39 patients with chronic sarcoidosis (duration greater than 4 years; 30 active, 9 inactive diseases) without therapy and correlated the monokine release with parameters of T-cell alveolitis and the course of the disease. The T4/T8 ratio was higher in the active than in the inactive group without reaching statistical significance. TNF alpha as well as IL-1 is spontaneously released by AM of the active group 2,099 +/- 518 pg/ml TNF alpha/10(6) cells/24 h and 8/13 (IL-1+/total) respectively. In the inactive group the AM release 375 +/- 246 pg/ml TNF alpha/10(6) cells/24 h which is in the range of the control and 1 out of 5 patients was IL-1-positive. There was no correlation between the monokine release and any parameter of T-cell alveolitis. These data support the hypothesis that the inflammatory process in chronic sarcoidosis is dominated by the activity of AM and that this activity determines the course of the disease.
Primary immune responses to tetanus toxoid (TT) and primary and secondary immune responses to a rabbit TT internal image bearing anti-idiotype antibody (Ab2 beta 1) inoculated in Freund's complete adjuvant (FCA), saline (SAL) or syntex adjuvant formulation vehicle (SAF) via the intraperitoneal or subcutaneous route, were examined in mice. High anti-TT antibody (Ab3) titres are reported although the titre and persistence of the antibody response varied according to the adjuvant used in the priming and challenge inocula of Ab2 beta 1. Mouse Ab3 antibodies were elicited in mice inoculated with rabbit Ab2 beta 1 antibodies which in turn were elicited by an inoculum of mouse monoclonal anti-TT Ab1 antibody. Ab3 was shown to be identical to Ab1 by immunoblot analysis. Primary and secondary immune responses elicited by rabbit Ab2 beta 1 antibody protected mice against a lethal dose of tetanus toxin.
The expression of activation molecules on peripheral-blood CD4 and CD8 T lymphocytes and the serum concentrations of two products of activated T lymphocytes [interferon-gamma (IFN-gamma) and soluble interleukin-2 receptor (sIL-2R)] were measured in patients with acute severe asthma (ASA) and controls. Significantly higher percentages of CD4+ cells from patients with ASA expressed IL-2R, HLA-DR and VLA-1 as compared to controls (p less than 0.01). In contrast, CD8+ cells from both asthmatics and controls did not express IL-2R and VLA-1, and their expression of HLA-DR in asthmatics was not increased. Serum concentrations of IFN-gamma and sIL-2R were significantly elevated in patients with ASA as compared to control groups (p less than 0.01). Concentrations decreased as the patients improved clinically following therapy. Significant correlations were observed between the improvements in airways obstruction and (1) the decreases in the percentages of peripheral-blood IL-2R+ T lymphocytes and (2) the decreases in serum concentrations of sIL-2R. These observations suggest that CD4 T lymphocyte activation is important in the pathogenesis of ASA.
A simple quantitative method to measure nasal secretion in guinea pigs is described. Nasal secretion was measured with a piece of cotton thread dyed with fluorescein at one end which was inserted into an anterior naris and kept there for 60 s. The stretch of color of a thread dyed with fluorescein was proportional to fluid volume and to increase in weight of a thread due to absorbed nasal secretion induced by nasal provocation. In addition, the stretch of color due to nasal secretion was associated with the score of rhinorrhea. Thus, it is considered that the amount of nasal secretion can be reflected to the length of the stretch of color. Each secretion on the ipsilateral and the contralateral sides induced by nasal provocation could be separately measured by this method. The amount of nasal secretion induced by allergen in passively sensitized guinea pigs could be reduced by pretreatment with ketotifen or flutropium. These results suggest that our method may serve as a quantitative test for nasal secretion in guinea pigs, which would be useful in the study of hypersecretory response in the allergic model or in evaluating the effect of antiallergic drugs on nasal allergy.
A long-term co-culture of mononuclear cells of human umbilical cord blood with mouse embryo-derived 3T3 fibroblasts resulted in the development of human mast cells. These mast cells are morphologically and functionally mature cells, containing 1.4-2.8 micrograms histamine per 10(6) cells and bear approximately 10(5) Fc epsilon RI per cell. The mast cells sensitized with human IgE released histamine upon challenge with anti-IgE. Electron-microscopic analysis of the cells showed that these cells were mature human mast cells, and clearly different from basophilic granulocytes. Most of the mast cells contained some granules with regular crystalline arrays and both tryptase and chymase, resembling human skin mast cells. When mononuclear cells of cord blood were seeded in a millicell insert which was placed on 3T3 fibroblasts monolayer, the number of mast cells developed in the millicell inserts was comparable to those developed in the co-culture of the same cord blood cells with 3T3 fibroblasts. Recent observations that mast cells developed in the presence of concentrated culture supernatants of 3T3 fibroblasts without fibroblasts feeder layers, confirmed that soluble factors released from 3T3 fibroblasts are essential and sufficient for the differentiation of human mast cell progenitors in vitro. Analysis of functional characteristics of cultured mast cells revealed that they respond to anti-IgE, Ca2+ ionophore A23187 and substance P for histamine release, but failed to respond to compound 48/80 and FMLP. Upon anti-IgE challenge, sensitized mast cells generated approximately 80 ng PGD2 per 10(6) cells, and approximately 50 ng of LTC4 per 10(6) cells but no detectable generation of LTB4.(ABSTRACT TRUNCATED AT 250 WORDS)
[125I]-cyanopindolol (ICYP) was used in a binding assay to compare the number and affinity of beta 2-adrenoceptor binding sites on circulating polymorphonuclear leukocytes (PMN) of 21 children with atopic dermatitis (AD) and 23 age-matched controls. We found no correlation between the beta 2-adrenoceptor density and the age of the children in either collective. Furthermore, the number and affinity of beta 2-adrenoceptor binding sites on PMN was neither influenced by sex nor severity of disease, serum IgE level or percentage of eosinophils in the differential blood count. There was a tendency towards a lower number of beta 2-adrenoceptors in AD subjects (1,429 +/- 587 receptors/PMN) compared to controls (1,753 +/- 596 receptors/PMN), but this difference was statistically not significant.
Mouse monoclonal (Ab1) anti-tetanus toxin/toxoid antibodies were used to raise Ab2 beta (tetanus toxin/toxoid internal image bearing) anti-idiotype antibodies in rabbits. Those rabbit serum antibodies (Ab2 beta) that did not bind to mouse serum proteins on an affinity column gave rise to an Ab3 anti-tetanus toxin/toxoid antibody response in mice. Rabbit serum antibodies that did bind to the affinity column, when eluted and used to inoculate mice also gave rise to an Ab3 anti-tetanus toxin/toxoid antibody response. It is suggested that one population of rabbit Ab2 beta anti-idiotype antibodies (unbound fraction) bears a partial or complete internal image of a tetanus epitope (Ab2 beta 1) while others (bound fraction) bear a complete or partial mirror image of a mouse immunoglobulin epitope as well (Ab2 beta 2).
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.
The in vitro release of endogenous and exogenous PgF2 alpha from plasma and serum proteins by aspirin and other analgesic drugs has been studied by RIA and equilibrium-dialysis techniques, respectively. Before aspirin addition, the mean plasma level of PgF2 alpha measured by RIA was significantly lower in aspirin-sensitive asthma (ASA) patients (11.3 +/- 6.5 pg/ml; n = 8) than in aspirin-tolerant asthma (ATA) patients (25.0 +/- 11.4 pg/ml; n = 21). After aspirin addition (50 micrograms/ml) the mean PgF2 alpha level detected in plasma by RIA was higher in ASA patients (97.6 +/- 5.5 pg/ml) than in ATA patients (66.9 +/- 4.5). The binding of [3H]PgF2 alpha to serum protein was significantly inhibited by NSAIDs but not by paracetamol (0.2-1.0 mM). These results implicate PgF2 alpha and the protein-binding property of analgesic drugs in the pathogenesis of aspirin-sensitive asthma.
The effects of a potent antiallergic drug, pemirolast (TBX), on activation of signal-transducing phospholipase C and A2, were examined in rat peritoneal mast cells. TBX at concentrations between 10(-7) and 10(-5) g/ml effectively inhibited degranulation in response to both antigen and compound 48/80. At the same concentrations the drug markedly suppressed the formation of 1,2-diacylglycerol and phosphatidic acid, and the decrease in phosphatidylinositol, suggesting the prevention of phospholipase C activation. The blockade of phospholipase A2 was suggested by the decrease in agonists-induced arachidonic acid liberation. These results indicate that TBX may exert its inhibitory effects on mast cells, at least in part, by preventing the activation of signal-transducing phospholipases.
