Isocyanate vapor-induced antigenicity of human albumin

BACKGROUND: The bioreactivity of isocyanate, a leading cause of occupational asthma, has led to uncertainty regarding the chemical's antigenicity and mechanisms that elicit immunopathology. OBJECTIVE: To understand better the biologically relevant antigenic forms of hexamethylene diisocyanate (HDI), commonly used in the auto body industry. METHODS: Human albumin was exposed to HDI vapors through a novel approach designed to model the air-liquid interface of the human airway. Vapor HDI-exposed albumin was characterized by electrophoresis, chemical substitution analysis, mass spectrometry, and serology studies on auto body shop workers (N=203) and HDI asthmatics (N=11). RESULTS: HDI vapors caused significant changes in the shape and/or charge of human albumin, which differed from albumin exposed to liquid phase HDI, with lower isocyanate substitution ratios and distinct electrophoretic mobility. Specific sites of vapor HDI conjugation to albumin were identified at His(247) and Lys(414). Vapor HDI-exposed albumin was specifically recognized by the humoral arm of the human immune system, with a strong dependence on albumin as the carrier. Vapor HDI-exposed albumin-specific IgG titers were significantly associated with HDI exposure (P=.001), and specific IgE was detectable in 55% (6/11) of isocyanate asthmatics versus 1.5% (3/203) of exposed healthy workers. Parallel studies using HDI-exposed albumin conjugates produced by previously published methods showed less significant associations of HDI-specific IgG and IgE with exposure and disease, respectively. CONCLUSION: HDI-albumin conjugates produced by novel vapor phase exposure methods may be more physiologically relevant than those produced by previously published methods and of greater utility in characterizing the immune responses associated with HDI exposure and asthma.     

Human gamma/delta T-cell proliferation and IFN-gamma production induced by hexamethylene diisocyanate

BACKGROUND: The human immune response to isocyanate, a leading cause of occupational asthma, remains incompletely characterized, including the cell types involved and the form of the chemical that acts as an antigen. OBJECTIVE: The purpose of this investigation was to characterize human T cells that respond to hexamethylene diisocyanate (HDI), an aliphatic isocyanate routinely used in the automobile body industry. METHODS: Human T-cell lines were generated and characterized from peripheral blood of HDI-exposed and HDI-unexposed subjects, using two different HDI antigens, HDI-conjugated albumin and HDI-exposed human airway epithelial cells (NCI-H292). Flow cytometry was used to characterize the phenotype of HDI-responsive T cells. ELISA and intracellular staining techniques were used to evaluate HDI-induced cytokine production. DNA sequence analysis of T-cell receptors was used to further define clonal populations of HDI-responsive T cells. RESULTS: HDI antigen preparations but not "mock exposed" control antigens lead to increased proliferation of specific cell types, CD3+CD4-CD8(dim) and/or CD3+CD4-CD8- cells, from HDI-exposed but not from HDI-unexposed subjects. These HDI-responsive T cells expressed unique oligoclonal gamma/delta rather than alpha/beta T-cell receptors, with characteristics suggestive of antigen-mediated selection and specificity. The HDI-stimulated gamma/delta T cells were associated with T(H)1-like cytokines and produce IFN-gamma but not IL-5 or IL-13. CONCLUSIONS: These data are the first to demonstrate that HDI can selectively stimulate gamma/delta T cells with the potential to modulate the human immune response to exposure.     

Mycoplasma pneumoniae infection and environmental tobacco smoke inhibit lung glutathione adaptive responses and increase oxidative stress

Chronic cigarette smoking evokes a lung glutathione (GSH) adaptive response that results in elevated GSH levels in the lung epithelial lining fluid (ELF). Currently, little is known about how the lung regulates or maintains steady-state levels of ELF GSH. Pathogens such as Mycoplasma pneumoniae can exacerbate airway inflammation and oxidative stress. The present study examined whether M. pneumoniae infections synergize with environmental tobacco smoke (ETS) to disrupt lung GSH adaptive responses. Mice were exposed separately and in combination to ETS and M. pneumoniae for 16 weeks. ETS exposure resulted in a doubling of ELF GSH levels, which was blocked in the M. pneumoniae-exposed mice. In addition, the ETS-plus-M. pneumoniae-exposed mice had elevated levels of oxidized glutathione (GSSG), resulting in a dramatic change in the ELF redox state that corresponded with an increase in lung tissue DNA oxidation. Similar findings were observed in human lung epithelial cells in vitro. Cells exposed separately or in combination to cigarette smoke extract and M. pneumoniae for 48 h had elevated apical levels of GSH compared to control cells, and these increases were blocked by M. pneumoniae and were also associated with increased cellular DNA oxidation. Further studies showed that M. pneumoniae exposure blocked ETS-induced increases in GSH reductase, an enzyme that recycles GSSG back to GSH, both in vitro and in vivo. These studies suggest that M. pneumoniae infection synergizes with ETS and suppresses the lung's ability to respond appropriately to environmental challenges leading to enhanced oxidative stress.     

Human innate immune responses to hexamethylene diisocyanate (HDI) and HDI–albumin conjugates

Background Isocyanates, a leading cause of occupational asthma, are known to induce adaptive immune responses; however, innate immune responses, which generally precede and regulate adaptive immunity, remain largely uncharacterized. Objective The aim of the study was to identify and characterize the cellular, molecular and systemic innate immune responses induced by hexamethylene diisocyanate (HDI). Methods Human peripheral blood mononuclear cells (PBMCs) were stimulated in vitro with HDI–albumin conjugates or control antigen, and changes in phenotype, gene and protein expression were characterized by flow cytometry, microarray, Western blot and ELISA. Cell uptake of isocyanate was visualized microscopically using HDI–albumin conjugates prepared with fluorescently labelled albumin. In vivo, human HDI exposure was performed via a specific inhalation challenge, and subsequent changes in PBMCs and serum proteins were measured by flow cytometry and ELISA. Genotypes were determined by PCR. Results Human monocytes take up HDI–albumin conjugates and undergo marked changes in morphology and gene/protein expression in vitro. The most significant (P‐values 0.007–0.05) changes in mircoarray gene expression were noted in lysosomal genes, especially peptidases and proton pumps involved in antigen processing. Chemokines that regulate monocyte/macrophage trafficking (MIF, MCP‐1) and pattern‐recognition receptors that bind chitin (chitinases) and oxidized low‐density lipoprotein (CD68) were also increased following isocyanate–albumin exposure. In vivo, HDI‐exposed subjects exhibited a drastic increase in the percentage of PBMCs with the same HDI–albumin responsive phenotype characterized in vitro (HLA‐DR⁺/CD11c⁺ with altered light scatter properties). An exposure‐dependent decrease (46±11%; P<0.015) in serum concentrations of chitinase 3‐like‐1 was also observed in individuals who lack the major (type 1) human chitinase (due to genetic polymorphism), but not in individuals possessing at least one functional chitinase‐1 allele. Conclusions Previously unrecognized innate immune responses to HDI and HDI–albumin conjugates could influence the clinical spectrum of exposure reactions.     

Glutathione reductase targeted to type II cells does not protect mice from hyperoxic lung injury

Exposure of the lung epithelium to reactive oxygen species without adequate antioxidant defenses leads to airway inflammation, and may contribute to lung injury. Glutathione peroxidase catalyzes the reduction of peroxides by oxidation of glutathione (GSH) to glutathione disulfide (GSSG), which can in turn be reduced by glutathione reductase (GR). Increased levels of GSSG have been shown to correlate negatively with outcome after oxidant exposure, and increased GR activity has been protective against hyperoxia in lung epithelial cells in vitro. We tested the hypothesis that increased GR expression targeted to type II alveolar epithelial cells would improve outcome in hyperoxia-induced lung injury. Human GR with a mitochondrial targeting sequence was targeted to mouse type II cells using the SPC promoter. Two transgenic lines were identified, with Line 2 having higher lung GR activities than Line 1. Both transgenic lines had lower lung GSSG levels and higher GSH/GSSG ratios than wild-type. Six-week-old wild-type and transgenic mice were exposed to greater than 95% O2 or room air (RA) for 84 hours. After exposure, Line 2 mice had higher right lung/body weight ratios and lavage protein concentrations than wild-type mice, and both lines 1 and 2 had lower GSSG levels than wild-type mice. These findings suggest that GSSG accumulation in the lung may not play a significant role in the development of hyperoxic lung injury, or that compensatory responses to unregulated GR expression render animals more susceptible to hyperoxic lung injury.     

Occupational asthma caused by a polymer but not the monomer of toluene diisocyanate (TDI)

Isocyanates are the most common cause of occupational asthma. Isocyanate monomers and prepolymers are widely used in the manufacture of polyurethane compounds. However, prepolymers are generating increasing interest because of their lower volatility. No distinction has yet been made between asthmatic reactions caused by the monomers and the prepolymers of isocyanates, and asthmatic reactions caused by one type of isocyanate but not the other type have not been reported. We describe two wood-roof maintenance workers who developed asthma after being exposed to a varnish containing a prepolymer of toluene diisocyanate (TDI) with only small amounts of the monomer. Specific inhalation-challenge tests with the TDI monomer did not elicit significant airway obstruction, whereas exposure to the varnish and to the purified TDI prepolymer induced late asthmatic reactions. Specific antibodies against TDI monomer human serum albumin and TDI prepolymer human serum albumin conjugates could not be demonstrated. These observations demonstrate that isocyanate prepolymers can cause occupational asthma and that asthmatic reactions caused by isocyanate prepolymers, but not to the corresponding monomer, can occur in some exposed workers.     

Occupational asthma caused by a prepolymer but not the monomer of toluene diisocyanate (TDI).

Isocyanates are the most common cause of occupational asthma. Isocyanate monomers and prepolymers are widely used in the manufacture of polyurethane compounds. However, prepolymers are generating increasing interest because of their lower volatility. No distinction has yet been made between asthmatic reactions caused by the monomers and the prepolymers of isocyanates, and asthmatic reactions caused by one type of isocyanate but not the other type have not been reported. We describe two wood-roof maintenance workers who developed asthma after being exposed to a varnish containing a prepolymer of toluene diisocyanate (TDI) with only small amounts of the monomer. Specific inhalation-challenge tests with the TDI monomer did not elicit significant airway obstruction, whereas exposure to the varnish and to the purified TDI prepolymer induced late asthmatic reactions. Specific antibodies against TDI monomer human serum albumin and TDI prepolymer human serum albumin conjugates could not be demonstrated. These observations demonstrate that isocyanate prepolymers can cause occupational asthma and that asthmatic reactions caused by isocyanate prepolymers, but not to the corresponding monomer, can occur in some exposed workers.     

IgG antibodies against polyisocyanates in car painters

A group of thirty car painters exposed to vapours and aerosols of paint containing prepolymer and monomer of hexametylene diisocyanate (HDI) was investigated. Specific antibodies against monomer HDI and prepolymerized HDI were analysed with RAST (IgE) and ELISA (IgG) assays after conjugation of the haptens with human serum albumin. There was no significant increase of serum IgG antibodies against HDI monomer, nor of specific IgE antibodies against HDI monomer or prepolymer. Specific IgG antibodies against prepolymerized HDI were significantly increased, as compared with non-exposed referents (medians 0.11 vs 0.03 absorbance (A)). Six car painters were found to have specific IgG antibodies of subclass 4 against HDI prepolymer, four also against HDI monomer. This shows an association between exposure and specific IgG antibodies. Thirteen subjects had suffered symptoms of rhinitis and/or conjunctivitis, and ten had symptoms from the bronchi (two asthma). There was no significant association between symptoms and levels of specific antibodies. Most of the symptoms were slight and unspecific, probably due to irritative effects of the exposure.     

RS Virus-Induced Inflammation and the Intracellular Glutathione Redox State in Cultured Human Airway Epithelial Cells

There is ample evidence that asthma is mediated by oxidative stress and that viral infection, which is associated with asthma onset and exacerbation in infants, acts as one type of oxidative stress. The goal of this study was to determine whether respiratory syncytial virus (RSV) induces oxidative stress in cultured A549 human airway epithelial cells and normal human bronchial epithelial cells (NHBE), and whether such RSV-induced oxidative stress can induce airway inflammation. To evaluate the direct effect of RSV infection as an oxidative stressor, the intracellular levels of reduced glutathione (GSH) or oxidized glutathione (GSSG) were measured. Their ratio (GSH/GSSG) was calculated to indicate intracellular oxidation–reduction (redox) status in A549 and NHBE. To evaluate the extent to which glutathione redox regulation affected cytokine/chemokine production, the effect of pretreatment with a reductive agent, glutathione monoethyl ester (GSH-OEt) and RSV-specific monoclonal antibody was thus studied. RSV acted as a potent oxidative stressor on the intracellular glutathione redox state in human airway epithelial cells, activating signals to increase the production of cytokine/chemokine. Pretreatment with GSH-OEt significantly suppressed RSV-induced time-dependent changes in the intracellular redox state, and also suppressed RSV-induced up-regulation of epithelial cell-derived IL-8, IL-6 and eotaxin production, as well as RSV-specific monoclonal antibody. RSV-induced oxidative stress is likely to contribute to the perpetuation and amplification of the inflammatory response. Therapeutic intervention against oxidative stress might therefore be beneficial as adjunctive therapies for respiratory disorders that are caused by an RSV infection.     

Does leukotriene affect intracellular glutathione redox state in cultured human airway epithelial cells?

Leukotrienes (LTs) are one of the most important mediators in the pathophysiology of asthma. We measured the intracellular amounts of reduced glutathione (GSH) and oxidized glutathione (GSSG) in cultured human airway epithelial cells. LTC 4 affects the GSH/GSSG ratio by activating signals to increase interleukin-8 (IL-8) production. Pretreatment with a reducing agent, glutathione monochrome ester (GSH-OEt), and with a leukotriene receptor antagonist, montelukast, significantly suppressed LTC(4)-induced time-dependent changes in the intracellular redox state, and also suppressed upregulation of IL-8 production by suppressing NF-kappaB activation. Our observations led to the hypothesis that LTC(4)-induced oxidative stress is likely to contribute to amplification of airway inflammation.     

Experimental gastrointestinal allergy enhances pulmonary responses to specific and unrelated allergens

BACKGROUND: Gastrointestinal allergy often precedes or coexists with respiratory allergy. OBJECTIVE: We hypothesized that established experimental gastrointestinal allergy would prime for the development of allergic respiratory responses. METHODS: BALB/c mice were sensitized with ovalbumin (OVA) in the presence of aluminum potassium sulfate and then subjected to intragastric saline or OVA challenges. After the development of allergen-induced gastrointestinal allergy, mice were intranasally exposed to either saline, OVA, or a neoaeroallergen house dust mite (HDM) extract. Airway inflammation (eg, bronchoalveolar lavage fluid cellularity, cytokine levels, and OVA-specific antibody levels) and airway responsiveness to methacholine exposure were assessed after intranasal allergen exposure. RESULTS: A single intranasal exposure to OVA induced significantly more airway inflammation in intragastric OVA-challenged mice compared with that seen in intragastric saline-treated mice. Kinetic analysis revealed that the observed amplification of lung inflammation was sustained for up to 12 days after the last intragastric OVA challenge after resolution of blood eosinophilia. When mice with gastrointestinal allergy were repeatedly challenged with HDM in the respiratory tract, they experienced enhanced airway inflammation, including bronchoalveolar lavage fluid eosinophilia and increased IL-13 levels. CONCLUSION: Taken together, our results demonstrate that OVA-induced gastrointestinal allergy enhances not only allergic airway responses to OVA but also to HDM, an unrelated aeroallergen. CLINICAL IMPLICATIONS: Experimental gastrointestinal allergy primes for responses to allergens in the respiratory tract, enhancing antigen-specific antibody and T(H)2 cytokine production, airway inflammation, and airway hyperresponsiveness.     

Glutathione and GSH-dependent enzymes in bronchoalveolar lavage fluid cells in response to ozone.

The purpose of this study was to determine if in vivo ozone exposure results in elevations in the levels of glutathione and glutathione-dependent enzymes in cells derived from bronchoalveolar lavage fluid (BALF). Our hypothesis was that, as part of a defense mechanism against oxygen toxicity, such cells would have increased levels of glutathione (GSH) in response to an oxidant stress. Female F344/N rats were exposed to 0.8 ppm ozone, 6 hr/day, for 1, 3, or 7 days, after which cells were collected by lung lavage. The GSH and GSH-peroxidase activity per milligram of protein in the cellular fraction, both necessary for reducing cellular peroxides, were elevated after 3 days of ozone exposure. After 7 days of exposure, cellular GSH had returned to control values, but the activity of glutathione reductase, the enzyme that reduces oxidized glutathione to GSH, was increased. Extracellular GSH concentration and glutathione reductase activity in BALF were also increased after 7 days of exposure. The total glutathione equivalents (GSH and GSSG, both cellular and extracellular) in BALF increased throughout the 7-day exposure, with GSH increasing first in the cells, and then in the extracellular fluid. This study demonstrated that the glutathione anti-oxidant system of BALF cells is stimulated by exposure to ozone. This response may serve to protect cells from the toxic effects of oxidant stress.     

Glutathione redox regulates airway hyperresponsiveness and airway inflammation in mice

Glutathione is the major intracellular redox buffer. We have shown that glutathione redox status, which is the balance between intracellular reduced (GSH) and oxidized (GSSG) glutathione, in antigen-presenting cells (APC) regulates the helper T cell type 1 (Th1)/Th2 balance due to the production of IL-12. Bronchial asthma is a typical Th2 disease. Th2 cells and Th2 cytokines are characteristic of asthma and trigger off an inflammation. Accordingly, we studied the effects of the intracellular glutathione redox status on airway hyperresponsiveness (AHR) and allergen-induced airway inflammation in a mouse model of asthma. We used gamma-Glutamylcysteinylethyl ester (gamma-GCE), which is a membrane-permeating GSH precursor, to elevate the intracellular GSH level and GSH/GSSG ratio of mice. In vitro, gamma-GCE pretreatment of human monocytic THP-1 cells elevated the GSH/GSSG ratio and enhanced IL-12(p70) production induced by LPS. In the mouse asthma model, intraperitoneal injection of gamma-GCE elevated the GSH/GSSG ratio of lung tissue and reduced AHR. gamma-GCE reduced levels of IL-4, IL-5, IL-10, and the chemokines eotaxin and RANTES (regulated on activation, normal T cell expressed and secreted) in bronchoalveolar lavage fluid, whereas it enhanced the production of IL-12 and IFN-gamma. Histologically, gamma-GCE suppressed eosinophils infiltration. Interestingly, we also found that gamma-GCE directly inhibited chemokine-induced eosinophil chemotaxis without affecting eotaxin receptor chemokine receptor 3 (CCR3) expressions. Taken together, these findings suggest that changing glutathione redox balance, increase in GSH level, and the GSH/GSSG ratio by gamma-GCE, ameliorate bronchial asthma by altering the Th1/Th2 imbalance through IL-12 production from APC and suppressing chemokine production and eosinophil migration itself.     

Immunotoxic Effects of Mercuric Compounds on Human Lymphocytes and Monocytes. IV. Alterations in Cellular Glutathione Content

The major goal of this investigation was to determine if the sensitivity of lymphocytes and monocytes to mercury (Hg⁺⁺) was related to intracellular glutathione² (GSH) levels and the thiol redox status [GSH/glutathione disulfide (GSSG)]. To isolate cells based upon their GSH content, T and B-cells were stained with monochlorobimane (MCB) and separated into high and low fluorescent groups by FACS analysis. Cells with high GSH fluorescence were found to be resistant to both the cytotoxic and immunotoxic effects of HgCl₂ as evidenced by cell viability and their responsiveness to mitogen, respectively. In contrast, cells with low levels of GSH were extremely sensitive to mercury. To further examine the relationship between GSH level and mercury exposure, T-cells, B-cells and monocytes were treated with different doses of HgCl₂ for 12 hrs. All cells exhibited a dose-dependent decrease in GSH content with a concomitant reduction in GSSG levels. However, the GSH/GSSG ratio in these cells remained constant, or increased following exposure to mercury. GSH levels were also reduced in monocytes following exposure to HgCl₂; in this case, GSSG levels remained constant and a decline in the GSH/GSSG ratio was observed. For all cell types, mercury did not inhibit the activities of GSH reductase and GSH peroxidase, enzymes responsible for oxidation/reduction of GSH and GSSG, respectively. Results of the study clearly show that susceptibility to the immunotoxic effects of HgCl₂ is, in part, dependent upon GSH levels and further that mercury inhibits GSH generation by lymphocytes and monocytes. Whether a decrease in GSH or a change in the thiol redox state causes functional deficits associated with low level mercury exposure remains to be determined.     

Cigarette smoke-induced differential regulation of glutathione metabolism in bronchial epithelial cells is balanced by an antioxidant tetrapeptide UPF1

Airway epithelium is a principal target for inhaled oxidants like cigarette smoke, which induce epithelial injury and thereby provoke pathogenesis of chronic airway diseases. Alterations in airway epithelial glutathione (GSH) metabolism are central in causing a loss of reducing environment, however, data are scarce on epithelial cells from larger bronchi. We showed a transient depletion of intracellular GSH in human bronchial epithelial cells after exposure to cigarette smoke condensate (CSC), which later followed by a prolonged elevation. Of the GSH-regulating enzymes, CSC increased mRNA expression of both catalytic (GCLC) and modifier (GCLM) subunits of glutamate-cysteine ligase. UPF1, a tetrapeptide GSH analogue, 4-methoxy-l-tyrosinyl-γ-l-glutamyl-l-cysteinyl-glycine, known to possess a 50-fold higher hydroxyl radical scavenging efficiency than does GSH, normalized the intracellular GSH level in the human bronchial epithelial cells under oxidative stress caused by CSC. UPF1 restored the GCLM and GSH reductase mRNA levels, which were significantly augmented by CSC treatment, back to the level of untreated control cells, referring to a successful establishment of control by UPF1 upon the over-accumulation of GSH. Moreover, UPF1 showed a significantly more potent antioxidant capacity than did N-acetyl-l-cysteine (NAC) and, compared to NAC, demonstrated a better potential to assure the whole GSH homeostasis in human bronchial epithelial cells.The current study suggests that UPF1 is capable of maintaining intracellular GSH level under CSC-induced oxidative stress in bronchial epithelial cells via balanced control over GSH-regulating enzymes, reflecting an improved perception of cellular redox conditions and thereby warranting improved adjustment of GSH accumulation.     

Reducing agents inhibit the contractile response of isolated guinea-pig main bronchi

BACKGROUND: Oxidants are involved in many respiratory disorders, including asthma and chronic obstructive pulmonary diseases. Reduced glutathione (GSH), one of the most important antioxidant compounds against oxidant free radicals, is particularly abundant in the respiratory epithelial lining fluid, where its concentration is increased in inflammatory disorders. OBJECTIVE: We hypothesized that reducing agents may have a direct effect on airway smooth muscle. Therefore, we studied the effects of GSH on airway smooth muscle contractility in guinea-pig main bronchi. In parallel, we evaluated superoxide anion generation associated with in vitro bronchial smooth muscle contraction. METHODS: Guinea-pig main bronchi were mounted in organ baths filled with Krebs-Henseleit solution. Concentration-response curves to acetylcholine (Ach) (10(-9)-10(-3) M), carbachol (10(-9)-10(-4) M), or histamine (10(-9)-10(-3) M) were performed in the presence or absence of either reduced or oxidized glutathione (GSSG) (10(-5)-10(-3) M). We also evaluated the effects of GSH and GSSG on allergen-induced contraction in main bronchi obtained from ovalbumin-sensitized guinea-pig. Superoxide dismutase (SOD)-inhibited cytochrome c reduction kinetics was performed to evaluate superoxide anion (O2-) production during Ach-induced contraction. RESULTS: Reduced but not oxidized glutathione significantly decreased smooth muscle contraction induced by Ach, carbachol, and histamine. Similarly, only the reduced form of glutathione attenuated the bronchoconstriction induced by allergen exposure in bronchi from sensitized animals. Finally, SOD-inhibited cytochrome c reduction kinetics demonstrated increased O2- production following bronchial smooth muscle contraction. This production was not affected by epithelium removal. CONCLUSION: Our findings show that GSH decreases bronchial smooth muscle contraction to different stimuli and that oxidant free radicals are produced during bronchial smooth muscle contraction. We suggest that oxidants are involved in the mechanisms of bronchoconstriction and that reducing agents could be a possible therapeutic option for airway obstruction sustained by bronchospasm.     

Differential roles for CD4 and CD8 T cells after diisocyanate sensitization: genetic control of TH2-induced lung inflammation

BACKGROUND: Exposure to diisocyanates is a major cause of occupational asthma. We previously developed a novel mouse model of diisocyanate-induced asthma involving epicutaneous sensitization to hexamethylene diisocyanate (HDI) that demonstrates many features of the human disease, including airway eosinophilia and mucus hypersecretion. OBJECTIVE: To determine what factors are critical for the development of HDI-induced airway inflammation, we investigated the strain distribution of this response and the roles of CD4(+) and CD8(+) T cells. METHODS: Mice were epicutaneously exposed to HDI and then challenged with HDI, either by means of inhalation to induce airway inflammation or on the ear to induce contact hypersensitivity (CHS). Lymph node cytokine production and serum antibodies were also measured. RESULTS: Induction of airway eosinophilia was highly dependent on the mouse strain used, with C57BL/6, A/J, CBA, C3H, and C57BL/10 mice all having significantly fewer eosinophils than BALB/c mice. HDI-specific antibodies and lymph node IL-5 and IL-13 production were also diminished in non-BALB/c strains. In contrast, CHS to HDI developed in all strains tested. Studies in mice deficient in either CD4(+) or CD8(+) T cells revealed that CD4(+) T cells were critical for HDI-induced airway eosinophilia, whereas CD8(+) T cells were the major effector cells in CHS. CONCLUSION: The data suggest that, in contrast to CHS, induction of T(H)2 responses after epicutaneous exposure to diisocyanates is strongly genetically influenced. Furthermore, the lung inflammatory response to inhaled HDI appears to depend primarily on effective generation of these CD4(+) T(H)2 responses, as is the case in atopic asthma.     

Effect of Ozone Exposure on Intracellular Glutathione Redox State in Cultured Human Airway Epithelial Cells

Intracellular oxidation and reduction (redox state) correspond closely to the surrounding environment. Most environmental factors affecting this balances such as oxidants, ultraviolet light, radioactive emissions, infections, and allergic reactions represent oxidative stress upon cells. We examined intracellular redox state after oxidative stress upon cultured human airway epithelial cells (Calu-3) by measuring reduced (GSH) or oxidized (GSSG) glutathione. We studied cytokine production, which is related to glutathione redox regulation, in response to ozone and also evaluated the effect of pretreatment with an ethyl ester of reduced glutathione (GSH-OEt) on cytokines. Ozone exposure (3.0 ppm, 3 min) time-dependently changed the redox state, while increasing production of interleukin(IL)-8 and IL-6, mRNA and protein. Treatment with GSH-OEt before ozone suppressed IL-8, but stimulated IL-6 production. Thus, oxidative stress affects intracellular glutathione redox state, in airway epithelial cells, activating signals to increase production of cytokine, modulation that may exacerbate respiratory symptoms.     

Immunotoxic Effects of Mercuric Compounds on Human Lymphocytes and Monocytes. IV. Alterations in Cellular Glutathione Content

Abstract

The major goal of this investigation was to determine if the sensitivity of lymphocytes and monocytes to mercury (Hg⁺⁺) was related to intracellular glutathione² (GSH) levels and the thiol redox status [GSH/glutathione disulfide (GSSG)]. To isolate cells based upon their GSH content, T and B-cells were stained with monochlorobimane (MCB) and separated into high and low fluorescent groups by FACS analysis. Cells with high GSH fluorescence were found to be resistant to both the cytotoxic and immunotoxic effects of HgCl₂ as evidenced by cell viability and their responsiveness to mitogen, respectively. In contrast, cells with low levels of GSH were extremely sensitive to mercury. To further examine the relationship between GSH level and mercury exposure, T-cells, B-cells and monocytes were treated with different doses of HgCl₂ for 12 hrs. All cells exhibited a dose-dependent decrease in GSH content with a concomitant reduction in GSSG levels. However, the GSH/GSSG ratio in these cells remained constant, or increased following exposure to mercury. GSH levels were also reduced in monocytes following exposure to HgCl₂; in this case, GSSG levels remained constant and a decline in the GSH/GSSG ratio was observed. For all cell types, mercury did not inhibit the activities of GSH reductase and GSH peroxidase, enzymes responsible for oxidation/reduction of GSH and GSSG, respectively. Results of the study clearly show that susceptibility to the immunotoxic effects of HgCl₂ is, in part, dependent upon GSH levels and further that mercury inhibits GSH generation by lymphocytes and monocytes. Whether a decrease in GSH or a change in the thiol redox state causes functional deficits associated with low level mercury exposure remains to be determined.     

A novel mouse model of diisocyanate-induced asthma showing allergic-type inflammation in the lung after inhaled antigen challenge

BACKGROUND: Exposure to diisocyanates, a group of highly reactive, low-molecular-weight compounds, is a major cause of occupational asthma. In contrast to mouse models of atopic asthma, previous mouse models of diisocyanate-induced asthma have failed to show lung inflammation with characteristics of human disease. OBJECTIVE: Our goal was to establish a novel mouse model of diisocyanate-induced asthma in which lung inflammation reminiscent of that seen in human asthma is generated after inhaled antigen challenge. METHODS: BALB/c mice were epicutaneously sensitized to hexamethylene diisocyanate (HDI) and then challenged with an HDI-protein conjugate administered by means of an intranasal droplet. RESULTS: HDI sensitization resulted in development of contact hypersensitivity and HDI-specific antibody production. Most importantly, however, vigorous inflammatory responses with characteristics of human asthma were generated in the lung after inhaled HDI challenge. Challenge of sensitized, but not unsensitized, mice resulted in airway eosinophilia, mucus hypersecretion, and production of T(H)1-type (IFN-gamma) and T(H)2-type (IL-4, IL-5, and IL-13) cytokines by lung inflammatory cells. Despite the mixed T(H)1/T(H)2 response induced by HDI sensitization, use of cytokine-deficient mice revealed that airway eosinophilia was mediated by T(H)2 cytokines and not by IFN-gamma. CONCLUSION: We report a novel mouse model of diisocyanate-induced asthma that, in contrast to previous models, demonstrates antigen-induced lung inflammation with characteristics of human disease. This model will allow investigation of the immunopathogenesis of diisocyanate-induced asthma and should provide insight into this common form of occupational disease.