Neutrophil activation in human inflammatory skin reactions

To determine the functional activity of neutrophils emigrating to the sites of ongoing human allergic inflammatory reactions, we assessed the intensity of their intracellular oxidative metabolism by measuring the intracellular oxidation by H2O2 of the dye, 2',7'-dichlorofluorescein diacetate (DCFH-DA), to fluorescent 2',7'-dichlorofluorescein. The intensity of intracellular fluorescence was measured by flow cytometry. In 13 atopic subjects, two denuded skin blister bases were challenged for 5 hours with pollen antigen and two blister bases with buffer. Both the level of histamine and mean number of cells recovered at antigen sites were greater than at buffer sites. Oxidation of DCFH-DA by neutrophils recovered from both antigen and buffer sites was significantly greater than that of autologous peripheral blood neutrophils (PBNs), reflecting an increase in the spontaneous generation H2O2 of cells recovered from the sites of inflammation. DCFH-DA oxidation by cells recovered from sites of antigen incubation was significantly greater than cells from buffer site incubations. Neutrophils from both antigen and buffer sites could be stimulated further in vitro by phorbol myristate acetate to the same level of H2O2 generation as phorbol myristate acetate-stimulated PBNs. In five subjects studied, the noncellular component of chamber fluids collected at 1 and 5 hours stimulated PBNs to increase their oxidative metabolism by 42% to 62%; however, this increased level of intracellular H2O2 was still much less than the spontaneous H2O2 generation observed in cells recovered from sites of allergic inflammation. The increased oxidative metabolism of neutrophils in human allergic and inflammatory skin reactions has important pathophysiologic implications for the role of these cells in inflammatory responses.     

Correlations of in vivo mediator release with late cutaneous allergic responses in humans : I. Kinetics of histamine release

To evaluate the contribution of mast cell-derived mediators in the late cutaneous allergic response, the duration and quantity of antigen-induced histamine release was compared to the intensity of the antigen-induced skin reactions in atopic volunteers. Chambers containing either pollen extract or buffer were appended to denuded bases for 1 hr and were replaced hourly with buffer for 3 additional hr. These were compared to the extinction dilution skin test titer and to the mean diameters of the 20-minute wheal and induration at 6 and 8 hr after intradermal injection of antigen. Chamber-fluid histamine levels were significantly higher at antigen than at buffer sites throughout the 4 hr. The hourly histamine levels correlated with the size of the induration at 6 and 8 hr but not with the wheal size or skin test titer. We conclude that (1) histamine is released for at least 4 hr at skin sites of antigen challenge as a consequence of prolonged release either from individual or sequentially activated mast cells, and (2) the quantity of histamine released correlates with the intensity of the late-phase skin response. We hypothesize that histamine might be a marker for prolonged release from the mast cell of other mediators that are responsible for the late-phase response.     

Cellular inflammatory responses in human allergic skin reactions

To define better the role of inflammation in the response to pollen antigens, we have used our skin chamber model to study inflammatory cells recovered from the sites of ongoing allergic reactions. In 15 atopic subjects, paired skin blister sites were simultaneously challenged with ragweed- or grass-pollen antigen or buffer for 5 hours. There were 10 times as many cells recovered at antigen (20.7 X 10(5)) than at buffer (2.0 X 10(5)) sites, p less than 0.005; greater than 97% of the cells recovered were neutrophils. The number of cells recovered at the antigen sites correlated with the total amount of histamine released (r = 0.57; p less than 0.05) but not with the extinction dilution skin test reactivity nor with the intensity of the late cutaneous allergic response measured 6 hours after the injection of antigen. Phase-contrast microscopic examination of the cells recovered from the antigen sites demonstrated that 82% to 95% were polarized compared to 0% to 1.5% of autologous blood neutrophils obtained simultaneously from the peripheral blood. Antigen site cells were as capable of serum-dependent phagocytosis as peripheral blood neutrophils. There was no significant difference in the migratory response to buffer, the chemoattractant N-formyl-methionyl-leucyl-phenylalanine, or leukotriene B4, but there was a significantly decreased response to platelet-activating factor when the cells recovered from antigen sites were compared to autologous blood neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early effects of corticosteroids on basophils, leukocyte histamine, and tissue histamine

The comparative effect in 11 atopic subjects of a single intravenous injection of methylprednisolone on sequential studies of blood eosinophils, basophils, leukocyte sensitivity to antigen for histamine release, leukocyte histamine content, and skin histamine was examined. No significant changes occurred in any parameter after placebo treatment. In contrast, 4 hr after intravenous treatment with steroid there were significant decreases in mean eosinophil counts (-95%), basophil counts (-72%), and histamine content of 1 X 10(7) leukocyte samples (-62%). Temporal changes in the latter paralleled alterations in circulating basophil levels. No significant changes occured in the antigen histamine release sensitivity, or the total skin histamine. Studies over a longer period after steroids in 4 subjects showed eosinophil and basophil levels at a nadir at 8 hr, remaining suppressed for 24 hr, and returned to pretreatment levels by 72 hr. Results suggest that corticosteroids induce a prominent decrease in leukocyte histamine due to a depletion of basophils without a decrease in histamine content per basophil, and that skin tissue histamine stores remain unchanged by such treatment.     

Interleukin-6 is released in the cutaneous response to allergen challenge in atopic individuals.

The mechanisms responsible for cutaneous response to antigen are complex. Interleukin-1 (IL-1) and interleukin-6 (IL-6) are proinflammatory cytokines that share many properties. Previous studies with a blister-chamber model have demonstrated IL-1 to be produced in the cutaneous response to antigen. Since IL-2 production by activated T cells and IL-6 production by macrophages are both stimulated by IL-1, we hypothesized that IL-2 and IL-6 may be involved in the cutaneous late-phase response (LPR) to antigen. We examined antigen-challenged sites for IL-2 immunoreactivity (ELISA) but found no difference between antigen- and diluent-challenged skin sites (N = 4). Since IL-2 has been demonstrated to be produced in response to IL-1 and IL-1 activity has been demonstrated to be greatest between hours 10 and 12, we speculated that IL-2 might not be detected until after hour 12. We were unable to demonstrate any increase in IL-2 production, even by extending our studies to 24 hours in two subjects. Antigen-challenged, skin blister-chamber fluids from atopic subjects demonstrated the appearance of IL-6 (ELISA) in pooled samples representing hours 1 1/2, 3 1/2, 5 1/2, and 7, but not at diluent control sites (p less than 0.05; N = 6). IL-6 reached a median peak of 0.66 ng/ml at 3 1/2 hours. Median levels of IL-6 fell to baseline at 8 hours, followed by a second peak of 0.25 ng/ml at hour 10. Three distinct patterns of IL-6 release were noted: early release of IL-6 followed by a sustained slower rise that peaked at hour 9 before declining to baseline levels at 12 hours, early release of IL-6 followed by a fall to baseline levels at hours 7 to 9 with a second smaller peak at hours 9 to 11, and isolated early release of IL-6. Early IL-6 production correlated with late histamine production (R = 0.801; p = 0.06), and late IL-6 production correlated with eosinophil influx (R = 0.813; p = 0.05). The area of the LPR at skin test sites correlated with early IL-6 peak levels (R = 0.977; p = 0.004) and with total early IL-6 production (R = 0.885; p = 0.05), but not with late IL-6 production.(ABSTRACT TRUNCATED AT 400 WORDS)     

Release of eosinophil granule proteins during IgE-mediated allergic skin reactions.

To determine whether the eosinophil (EOS), a prominent component of human allergic skin reactions, releases its potentially pathogenic components in vivo, we appended collection chambers to the bases of unroofed skin blisters and challenged the sites for varying time periods with either pollen antigen (Ag) or buffer (B)-control solutions. In seven sensitive subjects, continuous challenge with pollen Ag consistently induced release of more major basic protein (MBP) and eosinophil-derived neutrophil (EDN) than did B solution. Low levels of both MBP and EDN were observed during the first hour with increased accumulation during the second to fifth hour. Comparison of Ag- versus B-challenged site responses in individual subjects demonstrated significantly higher levels of both MBP and EDN at Ag than at B sites during the second to fifth hour. Levels of both MBP and EDN in the second to fifth hour correlated significantly with histamine release in the same sites in the first hour (r = 0.66 and 0.83, respectively). Imprints of the skin bases of the chambers after 5 hours demonstrated variable numbers of EOS at the Ag-challenged sites and only occasional EOS at the B-challenged sites; most cells on the skin bases were neutrophils. However, immunofluorescence localization of MBP in biopsy specimens of the blister bases revealed striking extra cellular MBP deposition. These findings indicate that EOS components accumulate in vivo in IgE-mediated human skin reactions, even when prominent EOS accumulation is not visualized, possibly because the EOS are degranulated in the allergic-reaction site. Release of EOS components in these reactions may be linked to earlier mast cell activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Release of histamine and tryptase during continuous and interrupted cutaneous challenge with allergen in humans.

To help in understanding the patterns of in vivo mediator release in human allergic skin reactions, we have used a skin chamber model to challenge the denuded bases of skin blisters of 11 sensitive subjects with pollen antigens (Ags) and codeine (C), a mast cell degranulator. Challenges were performed either (1) continuously for 6 hours or (2) in an intermittent fashion that is, Ag or C for the first hour, buffer for the next 4 hours, and then Ag or C during the sixth hour. Fluids in the overlying chamber were assayed for levels of the mast cell components, histamine and tryptase. There was peak release of both histamine and tryptase during the first hour of Ag incubation (89 +/- 11 ng/ml and 1428 +/- 260 ng/ml, respectively). At continuous Ag-challenge sites, there was a plateau of histamine levels (8.0 to 9.5 ng/ml) during the next 4 hours, whereas tryptase levels decreased progressively to baseline levels. Challenge of continuous Ag-incubation sites with C, a mast cell activator, led to another peak release of both histamine and tryptase. At interrupted Ag-challenge sites, histamine levels decreased abruptly, and tryptase levels decreased progressively after the first hour. Rechallenge of such sites with Ag during the sixth hour induced a peak release of histamine but no increase in tryptase levels. Continuous challenge with C for up to 5 hours in other sites induced an initial peak histamine release without a subsequent plateau. However, such a plateau of histamine (but not tryptase) release occurred after an initial C challenge if Ag was subsequently incubated in a continuous fashion.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Identification of IL-16 as the lymphocyte chemotactic activity in the bronchoalveolar lavage fluid of histamine-challenged asthmatic patients

Objective: We have previously demonstrated that the earliest lymphocyte chemotactic factors present in bronchoalveolar lavage fluid (BALF) of subjects with atopic asthma after subsegmental antigen challenge are IL-16 and MIP-1α, of which IL-16 appears to contribute a majority of the chemotactic activity. Because IL-16 is released in vitro after histamine stimulation of CD8⁺ T cells and epithelial cells, we evaluated the potential role of histamine in the release of IL-16 into the airways of allergic asthmatics in vivo. Methods: Eight allergic asthmatic subjects, six normal subjects, and six atopic nonasthmatic subjects were challenged with saline in the lingula and with serial concentrations of histamine (1 × 10^-7 to 5 × 10^-5 mol/L) in the right middle lobe followed by bronchoalveolar lavage (BAL) 15 minutes and 6 hours later. Results: The BALF from saline- and histamine-challenged lobes of normal subjects and atopic nonasthmatic subjects contained no significant lymphocyte chemoattractant activity. In six of the eight atopic asthmatic subjects, the histamine-challenged but not saline-challenged segment contained IL-16 chemotactic activity but no other identifiable lymphocyte chemoattractant activities at 6 hours. Conclusions: IL-16 appears in the airways after histamine challenge and therefore could contribute to the earliest infiltration of CD4⁺T cells and eosinophils observed after antigen challenge due to histamine release from mast cells. (J Allergy Clin Immunol 1998;101:786-792.)     

Comparison of plasma histamine and cyclic nucleotides after antigen and methacholine inhalation in man

Serial determinations of plasma histamine and cyclic nucleotides (adenosine monophosphate [AMP] and guanosine monophosphate [GMP]) were performed after inhalation of antigen and methacholine in four groups of subjects. In the first group, consisting of six antigen-sensitive subjects exhibiting bronchospasm after inhalation of ragweed or grass antigen, plasma histamine was elevated within 2 min and persisted for 30 min after inhalation of antigen. Peak histamine levels were between 18 to 80 ng/ml. In the second group, consisting of four nonatopic subjects, neither bronchospasm nor histamine was observed, despite inhalation of the same or 10-fold increased concentrations of antigen. In the third group, consisting of six subjects (three atopic and three nonatopic) exhibiting bronchospasm after inhalation of 2.5 to 10 mg of methacholine, sustained increases of histamine began at 1 min and persisted for 60 min after inhalation of methacholine. In the fourth group, seven subjects (two atopic, five nonatopic) without demonstrable bronchospasm despite inhalation of 2.5- to 10-fold increased doses of methacholine, no histamine was detected in the plasma at any time after inhalation of methacholine. Serial measurements of cyclic nucleotides showed no consistent changes in serum levels of cyclic AMP or cyclic GMP following inhalation challenge. We conclude that serum levels of histamine but not cyclic nucleotides change during bronchospasm induced by either antigen or methacholine.     

Late appearance of phospholipid platelet-activating factor and leukotriene B4 in human skin after repeated antigen challenge

Inflammatory mediators were assessed in supernatants of chamber fluids from eight ragweed- or grass-sensitive subjects during antigen-induced cutaneous inflammatory responses. Platelet activating factor (PAF) accumulated at concentrations of 1 pm to 90 mumol/L in six of eight subjects beginning at 3 hours and continuing for 9 hours after antigen challenge. Leukotriene B4 (LTB4) was detectable at cutaneous sites of antigen challenge in five of five subjects throughout the 9-hour period at levels from 1 to 36 nmol, a range of 38% to 80% of which were omega-oxidation metabolites. Histamine levels peaked in the first hour at 106 +/- 18 ng/ml and decreased to a plateau of 11 to 13 ng/ml at 3 to 9 hours after antigen challenge. No PAF and only very low levels of LTB4 (0.1 to 1.3 nmol) and of histamine (less than 2 ng/ml) were detected at buffer-control sites during the 9 hours of study. Continuous antigen exposure thus results in the persistent release of histamine and LTB4 and the late appearance of PAF, all of which may contribute to the chronicity of allergic disorders and may have a bearing on the IgE-mediated, late-phase cutaneous response.     

Potato lectin activates basophils and mast cells of atopic subjects by its interaction with core chitobiose of cell-bound non-specific immunoglobulin E

A major factor in non-allergic food hypersensitivity could be the interaction of dietary lectins with mast cells and basophils. Because immunoglobulin E (IgE) contains 10-12% carbohydrates, lectins can activate and degranulate these cells by cross-linking the glycans of cell-bound IgE. The present objective focuses on the effect of potato lectin (Solanum tuberosum agglutinin; STA) for its ability to release histamine from basophils in vitro and mast cells in vivo from non-atopic and atopic subjects. In this study, subjects were selected randomly based on case history and skin prick test responses with food, pollen and house dust mite extracts. Skin prick test (SPT) was performed with STA at 100 microg/ml concentration. Histamine release was performed using leucocytes from non-atopic and atopic subjects and rat peritoneal exudate cells. SPT on 110 atopic subjects using STA showed 39 subjects positive (35%); however, none showed STA-specific IgE; among 20 non-atopic subjects, none were positive by SPT. Maximal histamine release was found to be 65% in atopic subjects (n = 7) compared to 28% in non-atopic subjects (n = 5); the release was inhibited specifically by oligomers of N-acetylglucosamine and correlates well with serum total IgE levels (R(2) = 0.923). Binding of STA to N-linked glycoproteins (horseradish peroxidase, avidin and IgG) was positive by dot blot and binding assay. As potato lectin activates and degranulates both mast cells and basophils by interacting with the chitobiose core of IgE glycans, higher intake of potato may increase the clinical symptoms as a result of non-allergic food hypersensitivity in atopic subjects.     

Serum immunoglobulin levels in Australia antigen positive and Australia antigen negative hepatitis

Ig levels were determined by radial immunodiffusion in uncomplicated cases of acute hepatitis with or without Australia antigenaemia. Initial sera from Australia antigen negative cases showed a striking elevation in IgM levels when compared to Australia antigen positive cases (6·5 versus 1·9 mg/ml). None of twenty-four Australia antigen positive cases exceeded 3 mg/ml IgM, and only 3/58 Australia antigen negative cases exhibited values below 3 mg/ml. Intial sera from Australia antigen positive and Australia antigen negative subjects did not differ in concentration of IgG, IgA, or IgD. Serial determinations of IgG revealed a transient fall in patients with Australia antigen positive hepatitis, and a rise in Australia antigen negative cases. Asymptomatic, Australia antigen positive, Guaymi Indian subjects were compared to matched Australia antigen negative controls from the same indigenous group and no differences in the concentration of IgG, IgM, IgA or IgD were found, although elevations of IgG and IgM were common in both groups. No evidence of abnormal proteins was found when sera were tested by cellulose acetate electrophoresis or by immunoelectrophoresis versus immunoglobulin-specific antisera. Ultracentrifugal analysis failed to detect `7S' IgM.     

Bronchoalveolar lavage fluid mediator levels 5 minutes after allergen challenge in atopic subjects with asthma: relationship to the development of late asthmatic responses

Inflammatory mediators have been implicated in the pathogenesis of human asthma and have been demonstrated to increase in bronchoalveolar lavage fluid during the time of the immediate asthmatic response (IAR) after allergen instillation in the lungs. However, the relationship of these mediators, measured early to the late asthmatic response (LAR), airway reactivity, and clinical asthma, is unknown. In the present study, we evaluated mediator levels in bronchoalveolar lavage fluid before and 5 minutes after allergen challenge from three subject groups: atopic subjects without asthma (N = 7), atopic subjects with asthma and without LAR [-) LAR) (N = 6), and atopic subjects with asthma and with LAR [+) LAR) (N = 6). Subjects with asthma were differentiated into subjects with and without LARs based on at least a 15% decrease in FEV1 between 3 to 8 hours postallergen inhalation. The mediators, prostaglandin D2 thromboxane B2 leukotriene C4 (LTC4), and histamine, were measured both before and after allergen instillation. Baseline prechallenge levels were similar, except in the case of LTC4. LTC4 was detectable at baseline significantly more frequently in the atopic subjects with asthma with and without LAR when these subjects were compared to the atopic subjects without asthma (nine of 12 detectable versus one of seven detectable). In all groups, significant increases in mediator levels were observed in the groups with asthma postallergen challenge, compared to the atopic subjects without asthma. Atopic subjects with asthma and without LAR had significantly higher levels of all four mediators after challenge than atopic subjects with asthma and with LAR and atopic subjects without asthma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for histamine-mediated inhibition of monocyte chemotaxis in atopic dermatitis

Leukocyte chemotaxis was studied in 11 patients with severe childhood onset atopic dermatitis at a time when their disease was relatively quiescent. Pyoderma had been an important complication of the dermatitis in these patients. The chemotactic responsiveness of patient neutrophils and monocytes was on the average not significantly different from that of healthy control subjects, although three patients were identified who had significantly impaired responses. No correlation between IgE levels and leukocyte chemotaxis was observed. Because excessive amounts of histamine have been recovered from the skin of patients with atopic dermatitis, we evaluated the effects of histamine on the chemotactic responsiveness of leukocytes from these patients. Histamine caused a small dose-related increase in chemotaxis of neutrophils from both patients and control subjects (10^?7M to 10^?5M histamine). In contrast, histamine had no effect on the chemotaxis of monocytes from control subjects but inhibited the chemotactic responsiveness of monocytes from atopic dermatitis patients. These findings suggest that an abnormal sensitivity of monocytes to histamine is an intrinsic feature of atopic dermatitis that may be detectable when the disease is quiescent. Furthermore, this abnormality may contribute to the impairment of monocyte chemotaxis that has been previously observed in patients with active atopic dermatitis.     

Assessment of skin reactivity and specific antibody to egg and milk proteins in patients with atopic dermatitis: comparison with responses to the house dust mite antigen

Sixty patients with atopic dermatitis attending an allergy clinic were assessed for evidence of skin sensitivity and serum antibodies to egg and milk proteins. Prick skin test responses to egg were found in 23 patients and in 74% of these positive egg radioallergosorbent test (RAST) was demonstrable. Positive prick test for milk were present in 10 patients, but only 30% gave a positive milk RAST. Quantitative intradermal skin testing, RAST, and a double antibody antigen binding radioimmunoassay confirmed the presence of IgE antibody to egg proteins but indicated that the levels were very low when compared to those seen to the house dust mite antigen in sensitive patients. In contrast, IgG antibody to purified egg and milk proteins was present in large amount in most patients, the levels being significantly higher than in non-allergic controls.     

An in vivo demonstration of the antianaphylactic effect of terbutaline

Allergen-mediated histamine release was measured on small samples of blood in atopic subjects before and following ingestion of two tablets of terbutaline (5 mg) or placebo. No significant variation of histamine release was observed in the placebo group whereas a statistically significant decrease (maximally 45% of basal value) was found in four of the five patients receiving terbutaline. The mean reduction was about 25% of basal allergen-mediated histamine release. The inhibition was observed 1 hr after taking the drug and persisted for at least 5 hr. Twenty-four hours later the amount of histamine released by antigen was again at its basal value. These data indicate that terbutaline, at what are considered therapeutic doses, has an antianaphylactic action which might be of interest in the treatment of atopic disorders.     

Local histamine release after immunological and non-immunological mast cell degranulationin vivo

Plasma histamine concentration in the circulation has been proposed as an index of mast cell degranulation occurringin vivo but there are problems with this approach in practice. Local elevations in plasma histamine occur in blood draining the site of antigen challenge in forearm skin. We have compared changes in plasma histamine concentration with time following intradermal injection of antigen, codeine or histamine to produce matched wheal and flare responses in 4 atopic subjects. Less histamine appears to be released after non-immunological challenge.     

Patterns of mast cell alterations and in vivo mediator release in human allergic skin reactions

Patterns of in vivo histamine release in skin sites challenged with ragweed antigen were compared in five human subjects sensitive to this antigen and four nonallergic individuals, using a newly developed skin-chamber technique. These findings were compared with inflammatory cell responses in the reaction sites and patterns of ultramicroscopic mast cell alterations in biopsy specimens of skin tests in the same subjects. Definite mast cell alterations occurred within 15 sec and appeared maximal within 5 to 10 min after antigen injection. Histamine levels in appended chambers increased after a lag of 10 to 30 min and were elevated for at least 60 min after antigen challenge. Eosinophils accumulated only in antigen-induced reaction sites. However, there was no precise quantitative correlation among the degree of change in these three measurements. These appear to be promising approaches to further in vivo studies of human allergic reactions.     

The change of exhaled nitric oxide levels in early response after inhaled antigen in antigen-specific provocation test]

BACKGROUND: Inhaled antigen increases exhaled nitric oxide (eNO) in atopic asthmatics. Recent study showed that the increase of eNO levels was observed in late response (8-10 hours after inhaled antigen) but not in early response (1.5 hour after inhaled antigen). But we recognized that in some asthmatics eNO levels were increased during early response induced by antigen. METHODS: Atopic stable subjects with asthma induced by specific antigen (mite 11, housedust 3) were recruited in this study. Through bronchial provocation test with Mite or Housedust antigen, eNO levels were examined. As the control group, 7 atopic asthmatics who were not induced by specific antigen were recruited. RESULTS: In 7 subjects, the levels of eNO were increased during early response after inhaled antigen, and in other 7 subjects the levels of eNO were decreased. There were significant difference in the falling of FEV1 at threshold between the two groups (eNO increased group vs eNO decreased group, 22.1+/-0.87 (%) vs. 44.2+/-6.57 (%), p=0.016). In 6 subjects in control group, the levels of eNO were decreased. CONCLUSION: Inhaled antigen increased the levels of eNO in some asthmatics during early response in bronchial provocation test. The level of eNO has possibility of predicting the sudden decrease of FEV1 in bronchial provocation test.