Ovalbumin-induced airway inflammation and fibrosis in mice also exposed to ultrafine particles

A murine model of allergen-induced airway inflammation was used to examine the effects of exposure to ultrafine particles (PM(2.5)) on airway inflammation and remodeling. Lung inflammation was measured by quantitative differential evaluation of lung lavage cells. Alterations in lung structure (airway remodeling and fibrosis) were evaluated by quantitative biochemical analysis of microdissected airways and by histological evaluation of stained lung sections. The same total number of cells was observed in lavage fluid from animals exposed for 4 wk to ovalbumin alone or to ovalbumin for 4 wk immediately before or after 6 exposures over a period of 2 wk to 235 ug/m(3) of PM(2.5). Mice exposed to ovalbumin for 6 wk with concurrent exposure to PM(2.5) during wk 5-6 had a significant decrease in the total number of cells recovered by lavage as compared with the group exposed to ovalbumin alone. There were no significant differences in the cell differential counts in the lavage fluid from mice exposed to ovalbumin alone as compared with values from mice exposed to ovalbumin and PM(2.5) under the protocols studied. Airway structural changes (remodeling) were examined by three different quantitative methods. None of the groups exposed to ovalbumin and PM had a significant increase in airway collagen content evaluated biochemically (i.e., total airway collagen) as compared to the matched groups of mice exposed to ovalbumin alone. Airway collagen content evaluated histologically by sirius red staining showed significant increases in all of the animals exposed to ovalbumin, with or without PM, and no apparent difference between the ovalbumin group and mice exposed to PM with ovalbumin. The findings were consistent with an additive, or less than additive, response of mice to exposure to PM and ovalbumin. Air or PM exposure alone for 2 wk did not result in observable goblet cells in the airways, while mice exposed to ovalbumin aerosol alone for 4 wk had about 20-25% goblet cells in their conducting airways. Sequential exposure to ovalbumin and PM (or vice versa) caused significant increases in goblet cells (to about 35% of total cells) in the conducting airways of the exposed mice. We conclude that when mice with allergen-induced airway inflammation induced by ovalbumin are also exposed to PM(2.5), the lung inflammatory response and airway remodeling may be modified, but that this altered response is dependent upon the sequence of exposure and the duration of exposure to ovalbumin aerosol. At the concentrations of PM tested, we did not see changes in airway fibrosis or airway reactivity for animals exposed to ovalbumin and PM(2.5) as compared with animals exposed only to ovalbumin aerosol. However, goblet-cell hyperplasia was significantly increased in mice exposed concurrently to ovalbumin and PM(2.5) as compared with mice exposed to ovalbumin alone.     

Susceptibility to ovalbumin-induced airway inflammation and fibrosis in inducible nitric oxide synthetase-deficient mice: mechanisms and consequences

In a previous study, we showed that BALB/c mice demonstrate significant increases in accumulation of airway collagen after 4 weeks of exposure to ovalbumin aerosol. In the current study we examined the response to ovalbumin aerosol of a different strain of mice, C57BL/6, and compared this response to an otherwise isogenic C57BL strain (iNOS(-/-)) in which the gene for inducible nitric oxide synthetase (iNOS) had been knocked out. We hypothesized that C57BL mice, a Th-1-responsive strain, would be relatively resistant to ovalbumin exposure compared with our previous observations in the BALB/c strain, a Th-2 responder. Our results are consistent with this hypothesis, especially with respect to the accumulation of collagen in the airways of the mice exposed to ovalbumin and increased airway reactivity to challenge with methacholine, as measured by the Penh response. Since NO participates in multiple signal transduction pathways, there was no a priori reason to predict whether iNOS(-/-) mice would be more or less susceptible to allergen-induced airway inflammation than their parental wild-type strain. Responses to ovalbumin exposure of the Th-1-responsive C57BL animals were significantly less (or slower) than those we observed with the iNOS(-/-) mice. Significant increases in airway collagen content were seen only after 6 weeks of exposure of the C57BL mice, as contrasted with 4 weeks in the iNOS(-/-) animals. At each time point examined, Penh values for the iNOS(-/-) mice were significantly increased, while no increases were observed with the C57BL strain. Thus, the iNOS(-/-) mice are more susceptible to ovalbumin-induced airway inflammation and fibrosis than the C57BL strain, giving results intermediate between the previous observations in BALB/c mice and our current findings in C57BL animals with the various assays performed. We also asked whether the effects of knocking out the iNOS gene were exerted before or after the release of TGF-beta(1) by eosinophils and other effector cells in the lung. We measured the response of C57BL and iNOS(-/-) mice to direct intratracheal challenge with TGF-beta(1). There was no apparent response of C57BL mice to TGF-beta(1) at 4 or 11 days after TGF-beta(1) challenge, as evaluated by bronchoprovocation testing. On the other hand, the observed Penh values were significantly greater in iNOS(-/-) mice that had also received TGF-beta(1) 4 days previously. These results strongly support the hypothesis that the increased sensitivity of iNOS(-/-) mice to ovalbumin is at least partially dependent on pathways that come into play subsequent to the release of TGF-beta(1) by effector cells in the lungs of mice exposed to ovalbumin aerosol.     

Lung injury, inflammation, and inflammatory stimuli in rats exposed to ozone

The effects of ozone (O3) on airway epithelia, inflammation, and expression of inflammatory stimuli were investigated to delineate the mechanisms of inflammatory reactions relevant to lung injury. Because the airway responses to O3 develop gradually, this investigation included a time-sequence analysis. Rats exposed for 3 h to 1 ppm O3 were studied at 4-h intervals up to 20 h postexposure. Bronchoalveolar lavage fluid (BAL) was analyzed for albumin as an indicator of increased permeability, polymorphonuclear leukocytes (PMNs) to assess the inflammatory status, macrophage inflammatory protein-2 (MIP-2, an inflammatory chemokine), and cell adhesion molecules for their role in inflammation and PMN functions. The time-related increase in albumin was matched by a similar significant increase for PMNs, MIP-2, and intercellular adhesion molecule-1 (ICAM-1). However, no marked change occurred for beta-2 integrin (CD-18) and leukotriene B4 (LTB4). The results establish a temporal correlation of epithelial permeability with changes in inflammatory activity and stimuli responsible for PMN recruitment in the lung. The observations of elevated MIP-2 and ICAM-1 levels are consistent with their role in injury and inflammation. An early expression of MIP-2 mRNA in BAL cells, that is, immediately post O3 exposure, and the peak increase in BAL MIP-2 levels 4 h later support the chemotactic role of MIP-2 in PMN recruitment at 4- and 12-h time points. The rapid drop in MIP-2 and ICAM-1 levels appears to signal the termination of inflammatory cell recruitment, which is accompanied by an onset of recovery.     

The selective MMP-12 inhibitor, AS111793 reduces airway inflammation in mice exposed to cigarette smoke

BACKGROUND: Macrophage elastase (MMP-12) is involved in the inflammatory process of chronic obstructive pulmonary disease (COPD). The aim of this study was to investigate in mice the effect of MMP-12 inhibition on the inflammatory process induced by cigarette smoke (CS) or by lipopolysaccharide (LPS) exposure of the airways. EXPERIMENTAL APPROACH: C57BL/6 mice were given, orally, either the selective MMP-12 inhibitor AS111793 (3, 10, 30 and 100 mg kg(-1)), the PDE-4 inhibitor roflumilast (3 mg kg(-1)) or vehicle, then exposed to CS (for 3 days) or to LPS (100 microg mL(-1), 30 min). Subsequent to the last smoke or LPS exposure, bronchoalveolar lavages (BAL) were performed and lungs were removed and homogenized to analyze various markers of inflammation at appropriate times. KEY RESULTS: Inhibition of MMP-12 by AS111793 (10 and 30 mg kg(-1)) was associated with a reduction of the increase in neutrophil number in BAL fluids after 4 days and of macrophages after 11 days. On day 4, AS111793 also significantly reduced all the inflammation markers that had increased after CS exposure, including soluble TNF receptors I and II, MIP-1gamma, IL-6 and pro-MMP-9 activity in BAL fluids, and KC/CXCL1, fractalkine/CX3CL1, TIMP-1 and I-TAC/CXCL11 in lung parenchyma. In contrast, inhibition of MMP-12 did not reduce neutrophil influx, pro-MMP-9 activity or KC/CXCL1 release in BAL fluids of mice exposed to LPS. CONCLUSION: Inhibition of MMP-12 with AS111793, reduced the inflammatory process associated with exposure of mice to CS, strongly suggesting a specific involvement of MMP-12 in lung inflammation following CS exposure.     

Oxidative stress is an important component of airway inflammation in mice exposed to cigarette smoke or lipopolysaccharide

1. It was proposed previously that oxidative stress is a main component of the inflammatory process in chronic obstructive pulmonary disease (COPD). Thus, in the present study, we investigated the inflammatory response in mice deficient for the p47(phox) subunit of NADPH oxidase (p47 KO) exposed to cigarette smoke (CS). 2. Exposure of mice to CS elicited an increase in the number of macrophages and neutrophils and levels of interleukin (IL)-6, keratinocyte-derived chemokine (KC/CXCL1) and monocyte chemoattractant protein-1 (MCP1/CCL2) in bronchoalveolar lavage fluid (BALF), which were lower in p47 KO mice compared with control mice. In contrast, 24 h after lipopolysaccharide (LPS) exposure, the number of macrophages and neutrophils, as well as KC/CXCL1 levels, in BALF was significantly greater in p47 KO mice compared with control mice. 3. The present study has shown that airway inflammation is decreased in p47 KO mice after exposure to CS, but not LPS, suggesting that oxidative stress is involved in the pathogenesis of airway inflammation associated with COPD.     

Pulmonary inflammation in mice exposed to mainstream cigarette smoke

Male C57Bl/6 (C57) and ICR mice were exposed by nose-only inhalation to mainstream cigarette smoke (MS) from 2R4F reference cigarettes, at concentrations of 75, 250, and 600 microg of total particulate matter (TPM) per liter, for up to 6 mo. Respiratory-tract tissue (nose, larynx, and lung), blood, and bronchoalveolar lavage fluid (BALF) samples were collected and analyzed at several time points. Blood samples were analyzed for biomarkers of exposure (COHb and nicotine). BALF was analyzed for biomarkers of cell injury, inflammation, oxidative stress, enzyme activity, and cytokines. Blood COHb and plasma nicotine concentrations increased in a dose-dependent manner, confirming smoke exposure. Mild emphysema was observed following 28 wk of exposure. Macrophage accumulation and inflammatory infiltrates were observed around the alveolar ducts and adjacent vasculature. There was an approximately 13% increase in mean linear intercept (Lm) only in ICR mice exposed to 600 microg/L TPM. There were no significant changes in biomarkers of oxidative stress secondary to smoke exposures; however, 8-isoprostane significantly increased following the 13-wk post-inhalation period. BALF macrophage and neutrophil counts were rapidly and consistently elevated, while lymphocyte counts gradually increased over time. MS-induced inflammatory responses observed in this study are comparable to changes reported in chronic smokers, supporting the role of chronic inflammation in the pathogenesis of emphysema. However, mild emphysema in minimal numbers of mice suggests that MS exposure concentration and/or duration in the current study were not sufficient to induce a definitive emphysema phenotype.     

Enhanced antibody production in W/Wv mice exposed to ozone

To investigate the modulation of defense mechanisms by ozone (O3) exposure, mast-cell-deficient WBB6F1-W/WV and normal WBB6F1(-)+/+ mice were continuously exposed to 0.8 ppm O3 for 7 days. Although no differences in weights of lung, thymus and spleen were shown between exposed and control W/WV mice, antibody response to sheep red blood cells (SRBC) in exposed W/WV mice was markedly enhanced compared to control W/WV mice. In normal +/+ mice O3 exposure induced an increase in lung weight but did not enhance antibody production. These studies suggest that the susceptibility of W/WV mice to O3 may be different from that of +/+ mice.     

Airway fibrosis in a mouse model of airway inflammation

BALB/c mice were sensitized to ovalbumin by systemic injection and then exposed for up to 8 weeks to ovalbumin aerosols in whole body chambers. A pattern of airway inflammation, mucous cell hypertrophy and hyperplasia, and airway remodeling with submucosal fibrosis was observed as lesions evolved over time. Larger conducting airways were removed from the lungs by microdissection. Airway fibrosis was quantified by direct assay for collagen content, which was significantly increased after 4 and 8 weeks of exposure to ovalbumin aerosol. Based upon PCR analysis of mRNA levels in the airways, most of the newly synthesized collagen was Type I. Relaxin, administered by continuous infusion over the second half of a 4-week exposure to ovalbumin, was able to inhibit the accumulation of collagen in the airways of exposed mice. Thus, stimulation of collagen degradation by an activator of collagen breakdown by matrix metalloproteinases appears to be an effective therapeutic strategy in prevention of airway fibrosis in this animal model. Whole body plethysmography of unrestrained mice indicated functional changes in airway reactivity in the lungs of exposed animals occurring in conjunction with the reported structural changes. This result indicates that the ovalbumin-exposed mouse may be a suitable model for examining structure-function relationships in the lungs of animals with a predictable time course of airway inflammation, remodeling, and fibrosis and for testing potential new drugs for treatment of asthma or chronic bronchitis at a mechanistic level.     

Superoxide dismutase activity in lung from copper- and manganese-deficient mice exposed to ozone

Nutritional manganese (Mn) or copper (Cu) deficiency was investigated in Swiss-Webster mice exposed to ozone (O3). Mice rendered Mn-deficient were first reared from Mn-deficient dams and then fed a Mn-deficient (1 microgram/g) diet. Mice rendered Cu-deficient were fed a diet containing 0.2 microgram Cu/g diet. Control mice were fed a diet containing Mn at 45 micrograms/g and Cu at 8 micrograms/g. During the last week of the experiment (week 7, post-weanling), mice in each group were exposed continuously to 1.2 ppm O3 or filtered air for 7 days. Superoxide dismutase (SOD) activity in lung was then estimated. In mice breathing filtered air, neither lung Cu,Zn- nor Mn-SOD activity (U/g) was affected by diet. In O3-exposed mice, however, Mn-SOD activity was lower in the Mn-deficient group and Cu, Zn-SOD activity was lower in the Cu-deficient group. Moreover, total lung Cu,Zn-SOD activity was elevated in the Mn-deficient mice, whereas total Mn-SOD activity was elevated in the Cu-deficient mice in response to O3. These data indicate that under normal circumstances lung Cu,Zn-SOD and Mn-SOD are not affected by Cu or Mn deficiency. However, when an oxidant stress is superimposed on the Cu- or Mn-deficient condition, Cu,Zn- and Mn-SOD activities are impaired.     

Ovalbumin-induced neurogenic inflammation in the bladder of sensitized rats

1. We have developed and characterized a model of immediate hypersensitivity/inflammation of the urinary bladder in vivo induced by local application of ovalbumin (OA) in OA- sensitive female rats. Two parameters of the inflammatory response were assessed following OA challenge: plasma protein extravasation (PPE) and changes in smooth muscle reactivity. The former was estimated by measurement of Evans blue extravasation at 0.5, 2, 4, 8 and 24 h time point following in vivo challenge. Changes in reactivity were determined by measurement of isotonic tension responses of urinary bladder strips following OA challenge in vitro.
2. Acute in vivo intravesical OA challenge (10 mg in 0.3 ml saline) in actively sensitized female Wistar rats caused a time-dependent PPE in the urinary bladder which was biphasic with peak responses at 2–4 and 24 h.
3. The PPE response to acute OA challenge, above base-line, at 2 h was abolished by systemic capsaicin pretreatment (50 mg kg⁻¹, s.c., 4 days before use) (P<0.05) whilst the response at 24 h was unaffected. The 2 h time point was then used for further studies.
4. Degranulation of mast cells, achieved by pretreatment with compound 48/80 (5 mg kg⁻¹, s.c. for 3 consecutive days), completely abolished the PPE response to OA challenge at the 2 h time point.
5. The tachykinin NK₁ receptor antagonist, SR 140333 (0.1 μmol kg⁻¹, i.v.), abolished the 2 h PPE response whilst the tachykinin NK₂ receptor antagonist MEN 11420 (0.1 μmol kg⁻¹, i.v.) appeared to reduce the response by approximately 50% but this did not reach significance. The bradykinin B₂ receptor antagonist, Hoe 140 (0.1 μmol kg⁻¹, i.v.), similarly to SR 140333, blocked the 2 h PPE response to OA, whereas the selective B₁ receptor antagonist B 9858 (0.1 μmol kg⁻¹, i.v.) had no significant effect. Inhibition of cyclo-oxygenase (COX) achieved by pretreatment with the COX inhibitor dexketoprofen (5.3 μmol kg⁻¹, i.v.) also blocked the PPE response, whilst the leukotriene receptor antagonist ONO 1078 (1 μmol kg⁻¹, i.v.) significantly reduced PPE by about 80%.
6. In the rat isolated urinary bladder OA (1 mg ml⁻¹) challenge produced a biphasic response with a rapidly achieved maximal contraction followed by a sustained contraction for approximately 25 min. In vitro capsaicin pretreatment (10 μM for 15 min) significantly attenuated the duration of the sustained contraction whilst having no effect on the maximum contractile response achieved. In vivo pretreatment of animals with compound 48/80 significantly attenuated (42%) the maximum contractile response. Combination of both treatments almost completely abolished the response. In vitro treatment with Hoe 140 (1 μM) had no significant effect on the response to OA and neither did ONO 1078 (1 μM).
7. These results show that both the early inflammatory response and alterations in smooth muscle reactivity to OA challenge in actively sensitized animals are dependent on mast cell degranulation and the activation of sensory C-fibres. Furthermore this model of allergic cystitis may be useful for investigating both the processes involved and potential novel therapies in the treatment of interstitial cystitis.

     

Non-invasive biomarkers of pulmonary damage and inflammation: Application to children exposed to ozone and trichloramine

To date, airways injury or inflammation caused by air pollutants has been evaluated mainly by analysis of bronchoalveolar lavage, an invasive technique totally unsuitable to children. The assessment of respiratory risks in this particularly vulnerable population has thus for a long time relied on spirometric tests and self-reported symptoms which are relatively late and inaccurate indicators of lung damage. Research in the field of biomarkers is now opening new perspectives with the development of non-invasive tests allowing to monitor inflammation and damage in the deep lung. Blood tests measuring lung-specific proteins (pneumoproteins) such as Clara cell protein (CC16) and surfactant-associated proteins (A, B or D) are now available to evaluate the permeability and/or the cellular integrity of the pulmonary epithelium. The application of these tests to children has recently led to the discovery of a lung epithelium hyperpermeability caused by trichloramine (nitrogen trichloride), an irritant gas contaminating the air of indoor-chlorinated pools. Serum CC16 can also serve to detect increases of airway permeability during short-term exposures to ambient ozone. Indicators measurable in exhaled air such as nitric oxide (NO) appear more useful to detect airway inflammation. By applying the exhaled NO test to children attending summer camps, we recently found that ambient ozone produces an acute inflammatory response in children from levels slightly lower than current air quality guidelines. In a study exploring the links between atopy, asthma, and exposure to chlorination products in indoor pools, we also found that the exhaled NO test can serve to detect the chronic airway inflammation associated with excessive exposure to trichloramine. Lung-specific proteins measurable in serum and markers in exhaled air represent sensitive tools that can be used to assess non-invasively the effects of air pollutants on the respiratory tract of children.     

Microvascular remodelling in chronic airway inflammation in mice

1. Chronic inflammation is associated with blood vessel remodelling, including vessel proliferation and enlargement, and changes in vessel phenotype. We sought to characterize these changes in chronic airway inflammation and to determine whether corticosteroids that inhibit inflammation, such as dexamethasone, can also reduce microvascular remodelling. 2. Chronic airway inflammation was induced in C3H mice by infection with Mycoplasmapulmonis and the tracheal vessels treatment also decreased the immunoreactivity for P-selectin and the number of adherent leucocytes (595 +/- 203 vs 2,024 +/- 393 cells/ mm2 in treated and non-treated infected mice, respectively). 6. We conclude that microvascular enlargement and changes in vessel phenotype are features of some types of chronic inflammation and, furthermore, that dexamethasone reverses the microvascular enlargement, changes in vessel phenotype and leucocyte influx associated with chronic inflammatory airway disease.     

A latex-induced allergic airway inflammation model in mice

Latex allergy is important due to serious health impacts and widespread use of its products. Latex allergic reactions can be induced in skin and mucosal surfaces including the respiratory tract. The development of murine models of allergic airway inflammation has provided a framework to dissect out the cellular and molecular mechanisms of allergic respiratory inflammation. In this study we have developed a new mouse model of latex allergic airway inflammation using aerosol inhalation. The allergic inflammatory responses were characterized in this model. Mice were injected intraperitoneally with 0, 10, 50, or 200 microg of latex extract and their serum anti-latex IgE titers were determined. In the second stage, a standard protocol of inhalation was designed and three doses of latex extract solutions including 1%, 0.1%, and 0.01% were used to induce allergic airway inflammation. Bronchoalveolar lavage cytokines (IL-5 and IL-13) and serum anti-latex IgE and IgG(1) titers were determined by ELISA. Eosinophil levels in lung, peripheral blood, bronchoalveolar lavage and bone marrow were also evaluated. Histological analysis of lung tissue was also performed after latex inhalation. The aerosol inhalation of 1% latex allergens solution and presensitization with 50 mug of latex in this study resulted in the development of allergic airway inflammation characterized by elevated allergen specific IgE and IgG(1), peripheral blood, bronchoalveolar lavage and bone marrow eosinophilia. Histological analysis of the lung revealed an inflammatory response characterized by eosinophil accumulation. Elevated levels of Th2 cytokines IL-5 and IL-13 also were shown in bronchoalveolar lavage samples. These studies demonstrate that sensitization and subsequent aerosol inhalational challenge of latex allergen extract promotes allergic airway inflammation characterized by elevated IL-5 and IL-13 and eosinophils.     

Mepacrine treatment attenuates allergic airway remodeling segregated from airway inflammation in mice

Asthma is a chronic airway disease characterized by increased airway hyperresponsiveness, airway inflammation, and airway remodeling including collagen deposition in subepithelial regions. We have shown earlier that mepacrine has anti-inflammatory activity and decreased the features of airway remodeling in a subacute model of asthma, when administered during the inflammatory phase. But it was not clear whether the reduction of airway remodeling by mepacrine was a direct effect or indirectly related to the reduction in the airway inflammation. In this study, we determined the effect of mepacrine on airway remodeling and airway hyperresponsiveness (AHR) in a chronic model of asthma which showed the features of airway inflammation in the initial stage (inflammation predominant stage) and airway remodeling with mild airway inflammation in a later stage (remodeling predominant stage). Mepacrine was administered only in the later stage that more accurately simulates human asthma, where airway remodeling already exists at the time of diagnosis. The remodeling predominant stage was associated with high levels of Th2 cytokines like IL-4 and IL-13, increase in the levels of profibrotic mediators such as arginase and TGF-β, and increased collagen deposition. These were efficiently attenuated by mepacrine treatment and led to a significant reduction in AHR. Thus, we conclude from this study that mepacrine has direct effects on established airway remodeling independent of its anti-inflammatory effects.     

Baseline airway inflammation may be a determinant of the response to ozone exposure in asthmatic patients

Abstract

Context: It is well known that ozone exposure decreases lung function and increases airway neutrophilia, but large variability has been observed among asthmatic patients.

Objective: To find possible predictors of functional and inflammatory airway response to ozone in asthmatic patients.

Materials and methods: We studied 120 patients with mild-to-moderate asthma, randomly exposed to either air or ozone (0.3?ppm for 2?h) in a challenge chamber. Symptoms and pulmonary function test (PFT) were measured before and immediately after exposure. Six hours after exposure, induced sputum was collected. Patients were evaluated according to their functional (FEV₁ responders) or neutrophilic (neutrophil responders) response to ozone. We considered, as possible predictors of response: age, baseline FEV₁, previous treatment with inhaled corticosteroids (ICS), baseline sputum neutrophils, baseline sputum eosinophils, methacholine responsiveness, atopy and smoking habit.

Results: FEV₁ responders had lower baseline FEV₁, and a lower percentage of these had received ICS treatment. Neutrophil responders were younger, with lower baseline sputum inflammation and greater methacholine responsiveness. These results were confirmed by multivariate logistic analysis.

Discussion and conclusion: Patients not previously treated with ICS and patients with lower FEV₁ are more prone to functional response to ozone. Lower baseline airway inflammation and greater bronchial hyperresponsiveness may predict neutrophilic airway response to ozone in asthmatic patients. Thus, determinants of functional and inflammatory responses to ozone are different.     

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 g 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 g 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.