A low vitamin A status increases the susceptibility to cigarette smoke-induced lung emphysema in C57BL/6J mice.

Chronic obstructive pulmonary disease (COPD) is characterized by chronic airway inflammation. Cigarette smoke has been considered a major player in the pathogenesis of COPD. The inflamed airways of COPD patients contain several inflammatory cells. Vitamin A metabolites have been implicated in the repair of lung damage. Exposure to cigarette smoke has been shown to depress levels of retinol in lungs of rats. The purpose of this study was to investigate if a low, but not deficient, vitamin A status potentiated susceptibility to the development of cigarette smoke-induced lung emphysema in mice. Mice were bred that were the offspring's of 3 generations of mice that were fed a purified diet containing low levels of vitamin A and exposed to cigarette smoke for 3 months, every weekday. Then, levels of 9-cis, 13-cis, and all-trans retinoic acid, retinol and retinyl palmitate were measured in plasma, liver and right lung lobe. The left lung lobe was used to assess mean linear intercept (Lm), as a measure of smoke-induced lung damage. Average feed intakes were not different between treatment groups. We show that both retinol and retinyl palmitate levels were dramatically decreased in the storage organs of mice on the low vitamin A diet (retinol 2-fold in both lung and liver, and retinyl palmitate 5- fold in lung) which shows that the depletion was successful. However, this treatment did not result in the development of lung emphysema. However, smoke exposure led to a significant increase in Lm in mice with a low vitamin A status compared to the room air-breathing controls. Lung levels of acid retinoids were similar in all mice, irrespective of diet or smoke exposure. Concluding, a low vitamin A status increases the susceptibility to the development of cigarette smoke-induced lung emphysema, possibly because of decreased anti-oxidant capacity in the lungs due to locally reduced retinol and retinyl palmitate levels. These observations indicate that human populations with a low vitamin A status and a high prevalence of smoking may be at increased risk of developing lung emphysema.

A whey-based glutathione-enhancing diet decreases allergen-induced airway contraction in a guinea-pig model of asthma.

Since an allergen-induced early asthmatic reaction is likely to be accompanied by oxidative stress and since levels of the endogenous antioxidant glutathione can be enhanced by a whey-based diet (undenatured whey protein concentrate, UWPC), it was investigated whether UWPC could alleviate allergen-induced lung contractions. Guinea pigs were fed water or UWPC twice a day starting at day - 3 up to day 20. The animals were sensitised to ovalbumin or received saline on day 0. Serum samples were taken at several days after sensitisation to measure allergen-specific IgG. On day 20, lungs were isolated and perfused with buffer containing the allergen ovalbumin. Airway contractions were assessed, and mediators and indicators for oxidative stress were measured in the lung effluent. Moreover, glutathione levels were determined in the liver. The indicator of oxidative stress and airway contractile mediator, 8-iso-PGF(2α), was increased upon ovalbumin challenge in ovalbumin-sensitised groups. Furthermore, thiobarbituric acid-reactive substances (TBARS) were increased as well. Sensitisation with ovalbumin increased IgG levels from day 12 up to day 20, which were not influenced by the UWPC diet. In contrast, the UWPC diet significantly enhanced glutathione levels in the liver. Moreover, the UWPC diet significantly reduced the ovalbumin-induced anaphylactic response by 45 % and decreased PGE2 levels by 55 % in the effluent fluid. We show for the first time that during anaphylaxis, there is acute oxidative stress in the respiratory tract. The UWPC diet did not influence the sensitisation response to the allergen but did increase endogenous glutathione levels. The UWPC diet profoundly reduces allergen-induced airway constrictions, which opens new avenues for dietary management of allergic diseases.

Glutathione prevents the early asthmatic reaction and airway hyperresponsiveness in guinea pigs.

The prevalence of asthma has increased worldwide. The reasons for this rise remain unclear. Oxidative stress plays an important role in the pathogenesis of asthma. Glutathione (GSH) is the major representative of the class of nonprotein thiols and plays a pivotal role in a variety of enzymatic and nonenzymatic reactions that protect tissues against oxidative stress. In antioxidative reactions, GSH is converted into its oxidized form, glutathione disulfide (GSSG) that in its turn is enzymatically reduced into GSH to maintain a physiological redox balance. We used a guinea pig model of asthma to assess whether the early asthmatic reaction is associated with decreased lung levels of glutathione, and whether decreased glutathione is implicated in the increased airway smooth muscle reactivity that is associated with exposure of the lungs to allergen. Lung glutathione levels were decreased immediately after the onset of the early asthmatic reaction in vivo and associated with the release of 8-iso-PGF(2alpha), an indicator for oxidative stress. Glutathione ethylester, a glutathione precursor, blunted the airway obstruction during an early asthmatic reaction in a perfusion model and glutathione depletion rendered the airways hyperreactive. Glutathione ethyl ester in the buffer prevented this hyperreactivity. These results indicate that glutathione can modulate the early asthmatic reaction as well as the airway hyperresponsiveness.

Oral treatment with probiotics reduces allergic symptoms in ovalbumin-sensitized mice: a bacterial strain comparative study.

BACKGROUND/AIM: Evidence demonstrating an important role of the intestinal microbiota in the incidence of allergic disorders has led to the concept of using probiotics as possible antiallergic therapy. This study aimed to select a bacterial strain with the best antiallergic treatment effects from a panel of 6 bacterial strains in a mouse model of ovalbumin(OVA)-allergic asthma. METHODS: OVA-sensitized BALB/c mice were orally administered the bacterial strains Bifidobacterium breve M-16V, B. infantis NumRes251, B. animalis NumRes252 and NumRes253, Lactobacillus plantarum NumRes8 and L. rhamnosus NumRes6. After challenge by OVA inhalation in the lungs, the response to methacholine was measured. Pulmonary inflammation was assessed by analyzing bronchoalveolar lavage fluid for the presence of eosinophils, neutrophils, macrophages and lymphocytes and for interleukin 4, interleukin 5, interleukin 10 and interferon-gamma. OVA-specific IgE, IgG1 and IgG2a were measured in serum. Next, the effect on acute allergic skin reaction was measured after treatment with B. breve M-16V and L. plantarum NumRes8. RESULTS: Of the panel of 6 strains, B. breve M-16V and L. plantarum NumRes8 inhibited (1) the response to methacholine, (2) reduced the number of eosinophils in the bronchoalveolar lavage fluid, (3) reduced both OVA-specific IgE and (4) OVA-specific IgG1, whereas the other strains did not affect all these parameters simultaneously. B. breve M-16V but not L. plantarum NumRes8 reduced interleukin 4, interleukin 5 and interleukin 10. Furthermore, B. breve M-16V but not L. plantarum NumRes8 reduced acute allergic skin reactions to OVA. CONCLUSION: B. breve M-16V was identified as the most potent antiallergic strain.

Cigarette smoke induces CXCL8 production by human neutrophils via activation of TLR9 receptor.

Chronic obstructive pulmonary disease (COPD) is a major health problem and cigarette smoke is the main risk factor for the development of COPD. The characteristic changes in airway morphology, inflammatory cell infiltration and mediator expression in COPD may result from direct effects of cigarette smoke on airway cells. Toll-like receptors (TLRs) are key elements in pathogen recognition by the host immune system. Although TLRs have been intensely studied in innate immunity and infection, their critical role in noninfectious challenges has only recently emerged. Here we investigate whether cigarette smoke induces TLR9 signalling in human neutrophils. Human neutrophils were isolated from buffy coat and exposed to cigarette smoke extract. The production of CXC chemokine ligand (CXCL)8 was measured as a functional readout and the role of TLR9 signalling was investigated. Cigarette smoke extract induced CXCL8 release via TLR9 activation in neutrophils, which was confirmed in TLR9 stably transfected human embryonic kidney 293 cells. Moreover, cigarette smoke extract upregulated the expression of TLR9 and the upregulated expression was suppressed by N-acetylcysteine. TLR9 mediates cigarette smoke-induced release of CXCL8 and this may contribute to the accumulation of neutrophils and inflammation within the airways of smokers.

Cigarette smoke suppresses in vitro allergic activation of mouse mast cells.

BACKGROUND: Mast cells are important effector cells in innate or acquired immunity that contribute to host defence. Excessive activation of mast cells can result in the development of allergic diseases, including atopic asthma. Mast cell activation by IgE and specific antigen induces the cells to release spasmogenic, vasoactive and pro-inflammatory mediators, which enhance airway smooth muscle contraction, vascular permeability and inflammatory cell recruitment. Recently, we have demonstrated that exposure of mast cells to cigarette smoke medium (CSM) triggered mast cells to produce chemokines. On the other hand, smoking may decrease the risk of allergic sensitization, which could be explained by a reduced IgE production or a diminished response of mast cells to activation of the IgE receptor. OBJECTIVE: In this study, we investigated the effect of CSM on the allergic activation of mast cells through IgE and antigen. METHODS: Primary cultured murine mast cells were exposed to CSM and activated with IgE and antigen or lipopolysaccharide (LPS). The release of granules, production of leukotrienes, chemokines and cytokines was determined in the supernatants by ELISA. The effect of CSM exposure on intracellular signalling, especially the nuclear factor (NF)-kappaB and extracellular signal-regulated kinase (Erk)1/2 pathways, was analysed by Western blotting. RESULTS: CSM suppressed IgE-mediated degranulation and cytokine release, but no effect was observed on leukotriene release. CSM induced phosphorylation of Erk1/2 in mast cells. In CSM-exposed mast cells, activating transcription factor (ATF)-1 was phosphorylated after stimulation with IgE/Ag. LPS-activated mast cells were not influenced by CSM. CONCLUSION: Our study suggests that exposure to cigarette smoke may lead to a reduced allergic activation of mast cells without affecting their response to activation via e.g. bacterial-derived LPS.

Atopic and non-atopic allergic disorders: current insights into the possible involvement of free immunoglobulin light chains.

Allergic diseases have become a serious global health problem in the developed world. IgE interacting with its high-affinitiy receptor FcepsilonRI is considered a major contributing factor to most types of allergies, but depending on the type of allergy, however, a subgroup of patients displays common symptoms and yet lack elevated levels of total serum IgE and/or antigen-specific IgE. Novel therapeutic strategies such as anti-IgE therapy may therefore not be applicable to these patients. It is clear, however, that these patients do display activation of mast cells. In several patients suffering from immunological disorders, an increase in free immunoglobulin (IG) light chain levels can be detected. Previously, we have described the capability of free light chains to elicit immediate hypersensitivity responses. In this Opinion article, we will discuss the role of IgE- and non-IgE-mediated mechanisms in allergic disorders and point out a possible role of free IG light chains in the pathogenesis of the non-atopic types of these allergies.

Bacille-Calmette-Guerin vaccination and the development of allergic disease in children: a randomized, prospective, single-blind study.

BACKGROUND: The increase in the prevalence of allergic diseases in countries with a so-called western lifestyle may be due to a decrease in exposure to infectious agents in early life. OBJECTIVE: To establish the effect of Bacille-Calmette-Guerin (BCG) vaccination in 6-week-old high-risk infants in a prospective single-blind, randomized, placebo-controlled trial on the prevalence of allergic disease at the age of 4 and 18 months. METHODS: Subjects were 121 predominantly Caucasian high-risk newborns, having either a mother, or both a father and at least one sibling with past or present allergic disease. BCG or placebo was administered at the age of 6 weeks, and repeated once when both a post-vaccination scar and a positive TB skin test were absent at the age of 4 months. RESULTS: At the age of 18 months, the prevalence of allergic disease was not significantly different between the two groups. A trend towards less eczema (P=0.07) and significantly less use of medication for eczema was shown in the BCG group compared with the placebo group (P=0.04). CONCLUSION: A single (or once repeated) BCG vaccination in 6-week-old high-risk Caucasian infants was not associated with a 50% reduction in the prevalence of allergic disease. However, there could be a smaller beneficial effect of BCG, especially because a trend towards less eczema and significantly less use of medication for eczema was shown. For definite proof, a larger study should be carried out.

Dietary supplementation with specific oligosaccharide mixtures decreases parameters of allergic asthma in mice.

Specific mixtures of prebiotic oligosaccharides showed immune modulatory effects in previous murine vaccination experiments, suggesting a shift towards T-helper 1 (Th1) immunity. These mixtures consisted of short-chain galacto-oligosaccharides (scGOS) and long-chain fructo-oligosaccharides (lcFOS) in a 9:1 ratio (Immunofortis), with or without pectin-derived acidic oligosaccharides (pAOS). To investigate whether these mixtures could suppress Th2-related responses, they were tested in an ovalbumin (OVA)-induced model for experimental allergic asthma in BALB/c mice. Supplementation with two mixtures of scGOS/lcFOS and scGOS/lcFOS/pAOS at approximately 1% (w/w% net oligosaccharides) in the diet, starting two weeks before OVA sensitization and lasting until the end of the experiment, decreased of several parameters of allergic asthma. The OVA-induced airway inflammation and hyperresponsiveness was significantly suppressed by both mixtures. Moreover, OVA-specific IgE titers were decreased by more than 25%, although this effect was not significant. The effects of the oligosaccharide mixture with pAOS appeared to be more pronounced than the effects of the scGOS/lcFOS mixture without pAOS, but a direct comparison between the mixtures was not made. Overall, the results further support the hypothesis that specific mixtures of oligosaccharides modulate the Th1/Th2 balance by enhancing Th1-related and suppressing Th2-related parameters.

Topical application of F991, an immunoglobulin free light chain antagonist, prevents development of contact sensitivity in mice.

BACKGROUND: Exposure to reactive chemicals or environmental allergens can lead to hypersensitivity reactions in the skin of predisposed people. Most of these reactions are of atopic origin, but a subgroup of patients exhibits skin hypersensitivity reactions without features of atopy. OBJECTIVE: This study was undertaken to examine the effect of inhibiting the action of Ig-free light chains in a murine model for non-atopic skin hypersensitivity by dermal application of the free light chain antagonist F991. METHODS: To study the efficacy of F991, BALB/c mice were either passively immunized with trinitrophenyl (TNP)-specific immunoglobulin light chains (IgLC) and challenged with the hapten picryl chloride (PCl) or actively skin-sensitized and challenged with dinitrofluorobenzene (DNFB). The effect of F991 or control treatment was investigated by measuring local edema formation and the production of pro-inflammatory cytokines. RESULTS: Passive immunization with TNP-specific IgLC resulted in an increase in ear swelling 2 h after PCl challenge. F991 inhibited this enhanced ear swelling in a dose-dependent manner when applied 4 h before the sensitization with IgLC. F991 also inhibited DNFB-induced contact hypersensitivity reaction in the mouse skin 2 and 24 h after challenge when applied before challenge. Besides the prophylactic action, F991 when applied 2 h after DNFB-challenge, it was also able to attenuate symptoms of the DNFB-induced hypersensitivity reaction at 24 h after challenge. We showed that the beneficial effects of F991 are restricted to the side of application. CONCLUSION: F991 is able to effectively alleviate symptoms of contact sensitivity in mice. Our study suggests that local interference with IgLC-induced allergic symptoms may be attractive in the treatment of hypersensitivity responses.

Airway hyper-responsiveness in allergic asthma in guinea-pigs is mediated by nerve growth factor via the induction of substance P: a potential role for trkA.

BACKGROUND: The neurotrophin nerve growth factor (NGF) has been implicated as a mediator in allergic asthma. Direct evidence that inhibition of NGF-induced activation of neurotrophin receptors leads to improvement of airway symptoms is lacking. We therefore studied the effects of inhibitors of NGF signal transduction on the development of airway hyper-responsiveness (AHR) and pulmonary inflammation in a guinea-pig model for allergic asthma. METHODS: Airway responsiveness to the contractile agonist histamine was measured in vivo in guinea-pigs that were sensitized and challenged with ovalbumin (OVA). Inflammatory cell influx and NGF levels were determined in bronchoalveolar lavage fluid (BALF). Substance P, a key mediator of inflammation, was measured in lung tissue by radioimmunoassay, while substance P immunoreactive neurons in nodose ganglia were measured by immunohistochemistry. RESULTS: OVA challenge induced an AHR after 24 h in OVA-sensitized guinea-pigs. This coincided with an increase in the amount of NGF in BALF. Simultaneously, an increase in the percentage of substance P immunoreactive neurons in the nodose ganglia and an increase in the amount of substance P in lung tissue were found. We used tyrosine kinase inhibitors to block the signal transduction of the high-affinity NGF receptor, tyrosine kinase A (trkA). Treatment with the tyrosine kinase inhibitors (K252a or tyrphostin AG879) both inhibited the development of AHR, and prevented the increase in substance P in the nodose ganglia and lung tissue completely whereas both inhibitors had no effect on baseline airway resistance. Neither treatment with K252a or tyrphostin AG879 changed the influx of inflammatory cells in the BALF due to allergen challenge. CONCLUSIONS: We conclude that substance P plays a role in the induction of AHR in our model for allergic asthma which is most likely mediated by NGF. As both tyrosine kinase inhibitors AG879 and K252a show a similar inhibitory effect on airway function after allergen challenge, although both tyrosine kinase inhibitors exhibit different non-specific inhibitory effects on targets other than trkA tyrosine kinases, it is likely that the induction of substance P derived from sensory nerves is mediated by NGF via its high-affinity receptor trkA.

Stress and hypothermia in mice in a nose-only cigarette smoke exposure system.

In nose-only exposure systems, animals need to be restrained inside a tube, which leads to stress. Stress is known to cause hyperthermia in rodents. Chronically repeated episodes of hyperthermia could be detrimental to animal health and influence results of nose-only exposure studies. Therefore we investigated whether hyperthermia occurred in male C57BL/6J mice that were restrained for increasing lengths of time, using nosepieces held at room temperature, preheated at 37 degrees C, or thermostat controlled at different temperatures, with and without exposure to different concentrations of cigarette smoke. Body temperature, body weight, plasma corticosterone levels, and adrenal weights were recorded. Restraint using nosepieces at room temperature caused a time-dependent decrease in body temperature, which could be reversed by preheating the nosepieces to 37 degrees C. Cigarette smoke dose-dependently caused an additional decrease, which was counteracted by controlling nosepiece temperature at 38 degrees C. During 3 mo of exposure using heated nosepieces, Delta body temperature remained constant. Body weight gain did not differ between smoke-exposed and room air-breathing animals exposed using either heated or room-temperature nosepieces, but both groups gained significantly less weight, while adrenal weights were significantly and similarly increased, when compared to unrestrained littermates. Plasma corticosterone levels did not differ between the three groups. In conclusion, during restraint in nose-only exposure tubes with room temperature metal nosepieces, mice suffer a pronounced hypothermia. Preventing this by heating the nosepieces does not reduce the stress experienced by the animals.

Glutathione-conjugated toluene diisocyanate causes airway inflammation in sensitised mice.

Toluene diisocyanate (TDI) is a highly volatile compound that reacts readily with nucleophilic compounds, sulfhydryl groups in particular. Since the epithelial lining fluid of the airways contains high levels of the sulfhydryl, glutathione (GSH), inhalation of TDI is likely to result in the formation of GS-TDI conjugates. We therefore investigated whether GS-TDI is capable of provoking irritant and/or allergic reactions. Irritant effects of GS-TDI were studied after intratracheal administration of a range of doses of GS-TDI in saline to naive BALB/c mice. GS-TDI caused a dose-dependent increase in neutrophils in the lungs 24 h after instillation. A dose equivalent to 150 microg of TDI or lower had no effect. For provocation of allergic reactions, mice were sensitised by application of 1% TDI onto the skin on days 0 and 1, and challenged intratracheally with a sub-irritant dose of GS-TDI on day 8. GS-TDI did not induce non-specific tracheal hyperreactivity to carbachol 24 and 48 h after challenge in TDI-sensitised mice. However, it increased the numbers of neutrophils in the lungs as compared with the control mice. These findings suggest that GSH conjugation does not diminish the capacity of TDI to elicit irritant-induced inflammation in the lungs of mice at doses above 150 microg of TDI in the conjugate. Moreover, the capacity to induce allergic-specific inflammation was retained at concentrations of GS-TDI being devoid of irritant activity. However, the GS-TDI conjugate failed to induce non-specific tracheal hyperreactivity. This may be the consequence of the deposition of excess of GSH upon local dissociation of the conjugate.

CD28/CTLA4 double deficient mice demonstrate crucial role for B7 co-stimulation in the induction of allergic lower airways disease.

BACKGROUND: The existence of a third B7-1/B7-2 receptor was postulated in a recent study using a novel mouse strain lacking both CD28 and CTLA4 (CD28/CTLA4-/-). OBJECTIVE: In the present study, it was investigated if T cell co-stimulation via the putative B7-1/B7-2 receptor plays a role in the induction of Th2-mediated asthma manifestations in mice. METHODS: BALB/c wild-type, CD28/CTLA4-/- and B7-1/B7-2-/- mice were sensitized and aerosol challenged with ovalbumin (OVA). RESULTS: At 24 h after the last aerosol, wild-type mice showed airway hyper-responsiveness in vivo and up-regulated levels of serum OVA-specific IgE compared with the situation shortly before OVA challenge. In addition, eosinophil numbers and IL-5 levels in the broncho-alveolar lavage fluid and Th2 cytokine production by lung cells upon OVA re-stimulation in vitro were observed. In agreement with an earlier study, we failed to induce any of the asthma manifestations in B7-1/B7-2-/- mice. Importantly, also CD28/CTLA4-/- mice showed no asthma manifestations upon OVA sensitization and challenge. CONCLUSION: These data clearly demonstrate that T cell co-stimulation via the putative B7-1/B7-2 receptor appears to have no role in the induction of Th2-mediated asthma manifestations in this murine model and, conversely, that CD28 signalling is crucial.

Mycobacterium vaccae administration during allergen sensitization or challenge suppresses asthmatic features.

BACKGROUND AND OBJECTIVE: The hygiene hypothesis suggests that a lack of bacterial infections would favour the development of allergic disease. For this reason, bacteria or their components can be used as potential treatment for allergic asthma. We investigated whether heat-killed Mycobacterium vaccae is either able to suppress the induction of allergic asthma or able to suppress already established allergic asthma. METHODS: Mice were sensitized with ovalbumin (OVA)/alum on days 0 and 14. Thereafter, mice were challenged on days 35, 39 and 42 by inhalation of either OVA or saline aerosols. M. vaccae-treated mice received an injection with 106, 107 or 108 CFU heat-killed M. vaccae on days 0 and 14 or 107 CFU on days 35 and 39. On day 43, the airway responsiveness of the mice to increasing concentrations of methacholine was assessed, blood was withdrawn to measure serum parameters, and lung lavage was performed to detect cytokines and inflammatory cell number. RESULTS: Treatment of OVA-sensitized mice with 107 CFU M. vaccae either during sensitization or challenge suppresses airway hyper-responsiveness, airway eosinophilia and IL-5 production after OVA challenge. The increases in OVA-specific serum IgE and in IL-4 by respiratory challenges with OVA were only diminished after M. vaccae treatment (107 CFU) during sensitization. CONCLUSIONS: Heat-killed M. vaccae prevents allergic and asthmatic manifestations in a mouse model and, more importantly, M. vaccae treatment during challenge suppresses features of asthma, which opens up possibilities for new therapeutic interventions.

Modulation of airway hyperresponsiveness by thiols in a murine in vivo model of allergic asthma.

OBJECTIVE AND DESIGN: Since oxidative stress contributes to the pathogenesis of asthma, this study addressed the question whether supplementing the endogenous antioxidant, glutathione (GSH), would alleviate features of allergic asthma in the mouse. MATERIAL AND METHODS: Ovalbumin-sensitized mice received aerosols of the GSH-donors, glutathione-ethyl ester (GSEt) or N-acetylcysteine, before or during respiratory allergen challenges, or during methacholine challenges given one day after the last allergen challenge. Lung GSH levels were measured shortly after allergen or methacholine challenge. In addition, the effect of GSH supplements on airway hyperresponsiveness and inflammatory cell numbers in the airway lumen was assessed. RESULTS: GSEt decreased allergen-induced airway hyperresponsiveness when given in combination with methacholine. However, when given before or during allergen challenge, both GSH-donors failed to decrease the methacholine-induced airway contractility, change cell numbers in the airway lumen, or increase lung GSH levels. In addition, allergen challenges of sensitized mice did not decrease lung GSH levels. CONCLUSION: In contrast to guinea pigs and humans, allergen challenges in mice does not lead to acute oxidative stress.

Sodium cromoglycate and doxantrazole are oxygen radical scavengers.

The effects of two mast cell stabilisers, sodium cromoglycate (SCG) and doxantrazole, on the formation of reactive oxygen species (ROS) were studied. Guinea-pig alveolar macrophages (AMs) generated lucigenin-dependent chemiluminescence (LDCL). This was increased when the cells were stimulated by phorbol myristate acetate (PMA) or zymosan (by 133% and 464%, respectively, in total LDCL over 60 min). SCG decreased PMA-induced LDCL at higher concentrations (10 mM, by 55%) than doxantrazole (1 mM, by 75%). SCG decreased radical production by AMs in response to zymosan in a concentration-dependent manner by < or = 72%. Doxantrazole (0.1-1 mM) diminished total LDCL by 30-80%. In addition, glucose oxidase led to LDCL generation when incubated with glucose in a cell-free medium. This was inhibited by 47-83% in the presence of SCG or doxantrazole. SCG and doxantrazole inhibited the hydrogen peroxide- and peroxynitrite-induced LDCL by < or = 92%. Moreover, these drugs slightly increased the survival rate of the AMs. It is concluded that doxantrazole- and sodium cromoglycate-inhibited lucigenin-dependent chemiluminescence production by guinea-pig alveolar macrophages is due to a direct scavenging effect on reactive oxygen species. Doxantrazole is approximately 10-times more potent. Mast cell stabilisers may be effective in allergic asthma not only by preventing the allergen-induced mediator release, but also by preventing radical-induced lung damage.