Immunohistochemical characterization of the cellular infiltrate in airway mucosa of toluene diisocyanate (TDI)-induced asthma: comparison with allergic asthma.

Toluene diisocyanate (TDI) is the most prevalent agent in occupational asthma (OA) in Korea. The immuno-pathologic mechanism for TDI-induced bronchoconstriction remains to be clarified. We studied the immunohistochemical finding of inflammatory cells in bronchial mucosa in subjects with TDI-induced asthma. Fiberoptic bronchial biopsy specimens were obtained from nine subjects with TDI-induced asthma. Six allergic asthma sensitive to house dust mite were enrolled as controls. Bronchial biopsy specimens were examined by immunohistology with a panel of monoclonal antibodies to mast cell tryptase (AA1), secretary form of eosinophil cationic protein (EG2), pan T-lymphocyte (CD3) and neutrophil elastase (NE). There was a significant increase in the number of AA1+, EG2+ and NE+ cells in TDI-induced asthma compared to those of allergic asthma (p=0.02, p=0.04, p=0.03, respectively). No significant differences were observed in the number of CD3+ cells (p=0.27). These findings support the view that neutrophil recruitment together with eosinophil and mast cell, may contribute to the bronchoconstriction induced by TDI.     

Heterogeneity of IgE response to TDI-HSA conjugates by ELISA in toluene diisocyanate (TDI) -induced occupational asthma (OA) patients.

Toluene diisocyanate (TDI), a low molecular weight reactive chemical, is known to be a main cause of occupational asthma (OA) in Korea. Although it is thought that inhaled TDI may act as a hapten, the precise mechanisms of TDI-induced OA are unknown. In this study, TDI-human serum albumin (HSA) conjugates (5, 10, 20 and 30 min) were prepared in the range of 1.5 to 5.0 TDI mole/HSA mole. Specific binding of serum IgE to TDI-HSA (30 min) was observed using IgE ELISA as well as ELISA inhibition assay. Around 40% of TDI-induced OA patients were positive for serum specific IgE by ELISA. Degrees of serum IgE binding were different depending on which TDI-HSA conjugate was used as an antigen. Moreover, binding patterns were different depending on the individuals. Interestingly, higher binding of IgE to TDI-HSA (5 min) than to TDI-HSA (30 min) which was more highly substituted was observed in some patients. Probably new antigenic epitopes on carrier proteins were targets of the specific IgE. The results of this study indicated that IgE responses to TDI-HSA conjugates were heterogeneous in TDI-induced OA patients and self-proteins modified by reactive chemicals can become a major target antigen of IgE in certain cases.     

Cytokine secretion patterns of T cells responding to haptenized-human serum albumin in toluene diisocyanate (TDI)-induced asthma patients.

Our understanding immune response mechanisms to chemical allergens has been limited. It was partly due to the nature of antigens, recognized by T cells, not being well characterized. In the present study, we examined a hypothesis that a reactive chemical allergen, toluene diisocyanate (TDI), react with autologous proteins, thereby inducing T cell responses to the modified self protein in vivo. TDI-human serum albumin (HSA) conjugates were prepared and the presence of antigenic epitopes on the TDI-HSA conjugate was confirmed by IgE ELISA. We examined proliferative and cytokine production responses in TDI-induced asthma patients using the TDI-HSA conjugate as an antigen. Although proliferative responses of peripheral blood mononuclear cells (PBMCs) were not detected, production of IFN-gamma was observed in both PBMC and T cell lines obtained from some newly-diagnosed patients by ELISA. Mitogen-inducible IL-4 production was also detected in some T cell lines. Results of this study may have two implications. One is that presentation of haptenized-self protein to the immune system may induce activation of T cells. The other is that T cells responding to this modified self protein may play a role in the pathogenesis of the chemical allergen-induced asthma by producing cytokines such as IFN-gamma and possibly IL-4.     

Acute and chronic changes of vascular endothelial growth factor (VEGF) in induced sputum of toluene diisocyanate (TDI)-induced asthma patients

Vascular endothelial growth factor (VEGF) is a multi-functional cytokine involved in inflammation, repair and angiogenesis in asthmatic airway. This study aimed to evaluate the role of VEGF in immediate bronchoconstriction induced by TDI inhalation, and in chronic TDI-asthma patients. 11 newly diagnosed TDI-asthma patients (group I), 12 chronic TDI-asthma patients with persistent asthma symptoms followed for >4 yr and 15 unexposed healthy controls were enrolled. In group I, induced sputum and serum were collected before and 7 hr after placebo- and TDI-bronchoprovocation test (BPT). In group II, induced sputum and serum were collected every 2 yr. VEGF levels were measured by ELISA. There were no significant differences in sputum and serum VEGF levels between patients and controls. Before and after placebo and TDI-BPT, no significant changes were noted in sputum and serum VEGF levels of group I. In group II patients, sputum VEGF showed variable changes at 1-yr, then decreased significantly at 2-yr (p<0.05), while serum VEGF showed variable changes at 2-yr, which decreased significantly at 4-yr (p<0.05). These results suggest that VEGF may play a minor role in immediate bronchoconstriction after TDI-BPT. In chronic TDI-asthma, VEGF may be involved to 2 yr after the diagnosis and the contribution may decrease after then.     

Neutrophil chemotactic activity in milk-induced asthma

Four subjects with clinical histories of milk-induced asthma were studied (three allergic to cow's milk; one to soya milk). In each instance, skin prick tests, RAST (IgE and IgG₄), the basophil histamine release, and serum precipitins, using appropriate milk extracts, were negative. After the ingestion of milk all the subjects developed a reproducible and dose-dependent increase in airflow limitation. Three patients (two allergic to cow's milk; one to soya milk) gave a characteristic immediate-type reaction, which was maximal at 30 min after challenge. The fourth individual developed an isolated late-phase response, with maximal airways obstruction 3 hr after ingesting milk. In the three subjects who gave an early reaction, wheezing was accompanied by an elevation in circulating neutrophil chemotactic activity (NCA). This was not observed in the individual with the isolated late reaction. By Sephacryl S-400 gel-filtration chromatography it was shown that NCA of the early reactions eluted with proteins having an estimated molecular weight of 600,000 daltons. The immediate asthmatic response in peak expiratory flow rate and the elevation in NCA were inhibited by the prior oral administration of either disodium cromoglycate (DSCG) or oral beclomethasone dipropionate (BDP). In contrast, DSCG had no effect on airways obstruction in the subject with the isolated late asthmatic response, although inhibition was achieved by BDP.     

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.     

Combined asthma and alveolitis due to diphenylmethane diisocyanate (MDI) with demonstration of no crossed respiratory reactivity to toluene diisocyanate (TDI)

Two workers, who developed asthmatic symptoms, were studied with inhalation provocation tests using diphenylmethane diisocyanate (MDI) and toluene diisocyanate (TDI). The subjects showed specific asthmatic reactions to MDI challenge (more than 20% fall in FEV1), and one also had an alveolar response. Alveolitis was suggested by fever, basal crackles, increased neutrophil counts in venous blood and in bronchoalveolar lavage. The asthmatic reaction to MDI challenge was associated with an increase in airway responsiveness to methacholine in both subjects. We conclude that MDI is a cause of asthma and/or hypersensitivity pneumonitis in workers exposed to diphenylmethane diisocyanate.     

Expression of interleukin (IL)-4 and IL-5 proteins in asthma induced by toluene diisocyanate (TDI)

Background TDI-induced asthma exhibits clinical, functional and morphological similarities with allergen-induced asthma, suggesting that an immunological mechanism is involved in the sensitization to TDL In vitro studies using the technique of cloning lymphocytes demonstrated that a great proportion of T-cell clones derived from bronchial mucosa of subjects with TDI-induced asthma produced IL-5 and interferon-gamma, but not IL-4, upon in vitro stimulation. Objectives To investigate in vivo the role of IL-4 and IL-5 on the inflammatory response of the bronchial mucosa to TDI in sensitized subjects, we performed a quantitative analysis of bronchial biopsies. Methods We obtained bronchial biopsies from six subjects with TDI asthrha 48 h after an asthmatic reaction induced by TDI challenge (challenged group), in six subjects with TDI asthma 1–4 weeks after the last exposure to TDI (chronic group), and in six non-asthmatic controls. The number of eosinophils, mast cells, T-lymphocytes, and IL-4 and IL-5 protein positive cells was determined by immunohistochemistry in the area 100 μn beneath the epithelial basement membrane. Results The characteristic increase of submucosal eosinophils, but not of mast cells and T-lymphocytes, was observed in the subjects with TDI-induced asthma when compared with controls. No differences were detected between the two groups of asthmatics. In the subjects with TDI-induced asthma, cell immunoreactivity for IL-5 was increased when compared with normal controls. There was no difference in the expression of IL-5 protein between challenged and chronic asthmatics. In contrast, the expression of IL-4 protein was increased only in the asthmatic subjects tested after recent exposure to TDI. Conclusions We demonstrated that TDI asthma 48 h after specific bronchial challenge was associated with increased numbers of cells expressing IL-4 and IL-5, whereas chronic TDI asthma was associated with increased expression of IL-5, but not of IL-4. The results suggest that subjects who developed TDI asthma exhibit increased production of IL-5 even in the absence of a recent trigger by the exogenous sensitizer and that production of TH₂-like cytokines in TDI-induced asthma may not always be co-ordinately regulated in vivo.     

Increased sensitivity to toluene diisocyanate (TDI) in airways previously exposed to low doses of TDI

Repeated airway exposures to toluene diisocyanate (TDI) may cause sensitization and asthma. This study has examined the acute inflammatory response to TDI in guinea-pig tracheobronchial airways, the development of increased sensitivity to TDI and the effects of xanthines and a glucocorticoid on these responses to TDI. A restricted surface area of the tracheobronchial mucosa of Ketalar-Xylazin anaesthetized guinea-pigs was exposed to TDI, dissolved in olive oil, by means of 1 min infusions through an oral catheter. The TDI-induced inflammatory process was quantified by determination of airway luminal entry of plasma. Already 3 nl (approximately 20 pmol) of TDI produced a significant and sustained exudation response (P less than 0.001 to P less than 0.01, 5 and 17 hr after exposure). Pretreatment with intravenous enprofylline (25 mumol/kg) intraperitoneally or 26 mumol/kg by tracheal superfusion) was without effect. Two repeated exposures to TDI 3 nl (on days 1 and 8) made the animals hyperresponsive to TDI so that on day 15 a previously subthreshold dose of TDI (0.3 nl) produced significant exudation both at 5 and 17 hr after exposure (P less than 0.001 to P less than 0.01). Similarly, two repeated dermal exposures to a large dose of TDI (20 microliters) lowered the threshold for tracheal provocation with TDI. Budesonide (2.6 mumol/kg orally) given daily during the topical airway 'sensitization' regimen (days 1-14) significantly reduced the response to the subsequent 0.3 nl challenge dose of TDI (P less than 0.05). The effects of daily treatments with either theophylline (100 mumol/kg) or enprofylline (50 mumol/kg) were not significant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Toluene diisocyanate (TDI) regulates haem oxygenase-1/ferritin expression: implications for toluene diisocyanate-induced asthma

Diisocyanate is a leading cause of occupational asthma (OA). Diisocyanate‐induced OA is an inflammatory disease of the airways that is associated with airway remodelling. Although the pathogenic mechanisms are unclear, oxidative stress may be related to the pathogenesis of diisocyanate‐induced OA. In our previous report, we observed that the expression of ferritin light chain (FTL) was decreased in both of bronchoalveolar lavage fluid and serum of patients with diphenyl‐methane diisocyanate (MDI)‐induced OA compared to those of asymptomatic exposed controls and unexposed healthy controls. In this study of toluene diisocyanate (TDI)‐OA, we found identical findings with increased transferrin and decreased ferritin levels in the serum of patients with TDI‐OA. To elucidate whether diisocyanate suppresses FTL synthesis directly, we tested the effect of TDI on the FTL synthesis in A549 cells, a human airway epithelial cell line. We found that haem oxygenase‐1 as well as FTL was suppressed by treatment with TDI in dose‐ and time‐dependent manners. We also found that the synthesis of other anti‐oxidant proteins such as thioredoxin‐1, glutathione peroxidase, peroxiredoxin 1 and catalase were suppressed by TDI. Furthermore, TDI suppressed nuclear translocation of Nrf2 through suppressing the phosphorylation of mitogen‐activated protein kinases (MAPKs); extracellular‐regulated kinase 1/2 (ERK1/2); p38; and c‐Jun N‐terminal kinase (JNK). Peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) agonists, 15‐deoxy‐Δ^12,14‐PGJ2 and rosiglitazone rescued the effect of TDI on HO‐1/FTL expression. Collectively, our findings suggest that TDI suppressed HO‐1/FTL expression through the MAPK–Nrf2 signalling pathway, which may be involved in the pathogenesis of TDI‐induced OA. Therefore, elucidating these observations further should help to develop the therapeutic strategies of diisocyanate‐induced OA.     

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.     

Diverse profiles of specific IgE response to toluene diisocyanate (TDI)-human serum albumin conjugate in TDI-induced asthma patients

The prevalence studies on specific IgE to toluene diisocyanate (TDI)-human serum albumin (HSA) conjugate in TDI-induced asthma have shown variable results. In this study, we attempted to compare specific IgE bindings to TDI-HSA conjugate and its specificity using 3 different conjugates. Sera were collected from 20 TDI-induced asthma and 10 controls. Specific IgE were measured by ELISA using three TDI-HSA conjugates; two from Carnegie Mellon (CM; 98 and 99 CM conjugates) and one from Ajou University. To evaluate specificity and cross-reactivity, ELISA inhibition tests were applied. Positive and negative predictive values between Ajou conjugate and 98 CM conjugate were 75% and 100%. Those between Ajou and 99 CM were 100% and 93.8%. One patient showed an isolated positive response to the Ajou with negative responses to the other two conjugates. ELISA inhibition test using this patient's serum revealed the significant inhibitions by the Ajou and minimal inhibitions by the others. On the other hand, another patient showed an isolated positive response to 99 CM with negative responses to the others, and ELISA inhibition test showed significant inhibition by 99 CM with minimal inhibitions by the others. These results suggest that specific IgE bindings to a new antigenic determinant of TDI-HSA conjugate can be heterogeneous and differ from one individual to another.     

Inhalation challenge and pharmacologic studies of toluene diisocyanate (TDI)-sensitive workers.

Workers with "sensitivity" to toluene diisocyanate (TDI) studied in depth in an attempt to determine mechanisms of bronchial hyperreactivity. Tests included provocative inhalation challenge (PIC) with TDI and methacholine challenge. Blood samples obtained prior to and at various times after PIC were used to measure complement and split products of complement and plasma histamine levels and to determine dose-response slopes of lymphocyte cyclic adenosine monophosphate (cAMP) following stimulation with agonists. TDI-reactive individuals were all reactive to methacholine and responded to PIC with TDI by immediate, delayed, or dual bronchospastic reactions. No change in plasma histamine, total complement levels, or split products of complement were measurable. TDI reactors gave decreased lymphocyte cAMP dose response slopes to stimulation with isoproterenol, prostaglandin E1, and TDI, which suggests that impairment of adrenergic receptors may play an important role in TDI reactivity.     

Follow-up study of patients with respiratory disease due to toluene diisocyanate (TDI)

The outcome of the respiratory symptoms, pulmonary function tests and bronchial hyperresponsiveness was studied in forty-seven workers with respiratory disease due to toluene diisocyanate (TDI) (twenty-seven asthmatic and twenty non-asthmatic subjects) after about 2 years from the first examination. Eight of twelve asthmatic subjects who left the industry after the first examination complained at the follow-up of dyspnoea and wheezing, but pulmonary function tests were unchanged; bronchial hyperresponsiveness decreased in three, but most were still positive to challenge test with bethanechol at the follow-up. Fifteen subjects who continued their exposure to TDI showed at the follow-up a significant decrease of the spirometric parameters and an increase of the bronchial hyperresponsiveness, and symptoms of chronic bronchitis were more frequent at the second examination. Non-asthmatic subjects, both exposed and non-exposed to TDI at the second examination, showed a significant decrease of the pulmonary function tests but no relevant changes in bronchial hyperresponsiveness. Our data suggest that stopping occupational exposure to TDI frequently did not produce an improvement of the TDI bronchial asthma, and persistence of the occupational exposure causes a more rapid decline in the respiratory function.     

Neutrophil chemotactic factor of anaphylaxis (NCF-A) release in aspirin-induced asthma

Neutrophil chemotactic activity (NCA) following oral challenge with aspirin (ASA) was determined in ASA-intolerant asthmatic subjects, and in ASA-tolerant asthmatic and normal subjects. There was a statistically significant fall in FEV₁ and a rise in NCA (P <0.01) following challenge in the ASA-sensitive subjects compared with that of the ASA-tolerant subjects and normal controls. No significant difference was observed between the latter two groups. The chemotactic factor identified in this study had a molecular weight greater than 150 000 which is consistent with NCF-A (neutrophil chemotactic factor of anaphylaxis). The ASA-induced fall in FEV₁ and rise in NCA was further studied in three of the ASA-intolerant asthmatic subjects, with and without pretreatment with inhaled sodium cromoglycate. In these subjects, the drug inhibited both the oral ASA-induced bronchoconstriction and the increase in neutrophil chemotactic activity. These results suggest that ASA-induced asthma involves mediator release from mast cells, as shown by the increase in NCA following ASA challenge and the protective effect of sodium cromoglycate which is considered to inhibit mast-cell degranulation.     

Immunological investigation of individuals with toluene diisocyanate asthma

Thirteen subjects who experienced only the expected irritant respiratory and ocular effects from occupational exposure to toluene diisocyanate (TDI) were compared with eight workers who appeared clinically to be sensitized to this substance, as manifested by the prompt development of asthmatic symptoms on exposure to minute concentrations of TDI. In an attempt to document an immunological response to TDI in the latter group, several conjugates of TDI with human serum albumin were prepared. Gel diffusion, leucocyte histamine release, PCA in guinea-pigs, passive haemagglutination, passive transfer (P-K) and a few direct skin tests all failed to show antibodies to TDI. In lymphocyte culture, however, TDI–human serum albumin complexes produced stimulation of lymphocytes from seven of the eight subjects suspected of being sensitized and none of the controls.     

Role of tumor necrosis factor in toluene diisocyanate asthma

Nearly 9 million workers are exposed to chemical agents associated with occupational asthma, with isocyanates representing the chemical class most responsible. Isocyanate-induced asthma has been difficult to diagnose and control, in part because the biologic mechanisms responsible for the disease and the determinants of exposure have not been well defined. Isocyanate-induced asthma is characterized by airway inflammation, and we hypothesized that inflammation is a prerequisite of isocyanate-induced asthma, with tumor necrosis factor (TNF)-alpha being critical to this process. To explore this hypothesis, wild-type mice, athymic mice, TNF-alpha receptor knockout (TNFR), and anti-TNF-alpha antibody-treated mice were sensitized by subcutaneous injection (20 micro l on Day 1; 5 micro l, Days 4 and 11), and challenged 7 d later by inhalation (100 ppb; Days 20, 22, and 24) with toluene diisocyanate (TDI). Airway inflammation, goblet cell metaplasia, epithelial cell damage, and nonspecific airway reactivity to methacholine challenge, measured 24 h following the last challenge, were reduced to baseline levels in TNF-alpha null mice and athymic mice. TNF-alpha deficiency also markedly abrogated TDI-induced Th2 cytokines in airway tissues, indicating a role in the development of Th2 responses. Despite abrogation of all indicators of asthma pathology, TNF-alpha neutralization had no effect on serum IgE levels or IgG-specific TDI antibodies, suggesting the lack of importance of a humoral response in the manifestation of TDI-induced asthma. Instillation studies with fluorescein-conjugated isothiocyanate and TDI suggested that TNF-alpha deficiency also resulted in a significant reduction in the migration of airway dendritic cells to the draining lymph nodes. Taken together, these results suggest that, unlike protein antigens, TNF-alpha has multiple and central roles in TDI-induced asthma, influencing both nonspecific inflammatory processes and specific immune events.     

Importance of inflammatory and immune components in a mouse model of airway reactivity to toluene diisocyanate (TDI)

BACKGROUND: Nearly 9 million individuals are exposed to agents in the workplace associated with asthma, and isocyanates represent the most common cause of occupationally induced asthma. OBJECTIVES: Nonetheless, the immunological mechanisms responsible for isocyanate-induced asthma are not clear. A murine model for toluene diisocyanate (TDI) asthma is described and employed to examine inflammatory and immune components that may be involved in the disease. METHODS: Groups (n = 6) of C57BL/6J and athymic mice were sensitized by subcutaneous injection (20 microl on day 1, 5 microl on days 4 and 11), and 7 days later challenged by inhalation (100 p.p.b., days 20, 22 and 24) with TDI. Twenty-four hours following the last challenge the tracheae and lungs were examined for histological changes as well as for the expression of Th1, Th2 and pro-inflammatory cytokines. Mice were also examined for airway reactivity to methacholine challenge and for specific and total IgE and IgG antibodies. RESULTS: TDI sensitization resulted in increased reactivity to methacholine challenge as well as a significant inflammatory response in the trachea and nares of wild-type mice, but not in the athymic mice nor in the lungs of the C57BL/6J mice. Airway inflammation was characterized by inflammatory cell influx, goblet cell metaplasia and epithelial damage. Histological changes in the trachea were accompanied by increased mRNA expression of interleukin (IL)-4, tumour necrosis factor alpha, lymphotoxin beta, lymphotactin and Rantes, as well as TDI-specific IgG antibodies and elevated levels of total IgE. IgE-specific antibodies were not detected with this exposure regimen but were produced when the TDI concentrations were increased. CONCLUSIONS: These studies provide a unique murine model for occupational asthma that generates both inflammatory and immune mediators similar to those occurring in TDI-induced asthma in humans.