Serum ferritin and transferrin levels as serologic markers of methylene diphenyl diisocyanate–induced occupational asthma

Article Outline

• Abstract
• Methods
  • Study subjects
  • Skin prick test and bronchial challenge testing
  • Bronchoscopy and bronchoalveolar lavage
  • Two-dimensional electrophoresis and image analysis
  • In-gel digestion and mass spectrometric analysis
  • Measurement of ferritin and transferrin in sera
  • Statistical analyses
• Results
  • Characteristics of the study subjects
  • 2-DE analysis of BALF
  • Identification of the proteins by MALDI-TOF MS
  • Measurement of the serum ferritin and transferrin levels by ELISA
  • Determination of the optimal cutoff levels for serum ferritin and transferrin
• Discussion
• Acknowledgment
• References
• Copyright

Background

Although methylene diphenyl diisocyanate (MDI) may induce occupational asthma in the workplace, the pathogenic mechanisms are unclear.

Objectives

By using bronchoalveolar lavage fluid, we sought to identify proteins that were differentially expressed between subjects with MDI-induced occupational asthma (MDI-OA) and asymptomatic exposed controls (AECs).

Methods

To find proteins that were differentially expressed between the MDI-OA and AEC groups, 2-dimensional electrophoresis was performed by using bronchoalveolar lavage fluid obtained from subjects after MDI-specific inhalation challenge. The selected protein spots were then identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The clinical relevance of the differentially expressed spots was compared by ELISA using sera from the MDI-OA/eosinophilic bronchitis, AEC, and unexposed healthy control groups. Receiver operating characteristic curves were then plotted, and the sensitivity and specificity were determined.

Results

Twenty-three protein spots were identified that distinguished the subjects with MDI-OA from those in the AEC group. Among them, ferritin expression was downregulated whereas transferrin expression was upregulated in subjects with MDI-OA compared with AEC; these results were validated by ELISA using sera from the MDI-OA/EB and AEC groups. To identify subjects with MDI-OA, the optimal serum cutoff levels were 69.84 ng/mL for ferritin and 2.48 μg/mL for transferrin. When these 2 parameters were combined, the sensitivity was 71.43% and the specificity was 85.71%.

Conclusion

Serum ferritin and transferrin levels are associated with the phenotype of MDI-OA.

Key words: MDI, occupational asthma, ferritin, transferrin, proteome

Abbreviations used: 2-DE, 2-Dimensional electrophoresis, AEC, Asymptomatic exposed control, ANCOVA, Analysis of covariance, ARDS, Acute respiratory distress syndrome, AUC, Area under the curve, BALF, Bronchoalveolar lavage fluid, DBP, Vitamin D–binding protein precursor, EB, Eosinophilic bronchitis, MALDI-TOF/TOF MS, Matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry, MDI, Methylene diisocyanate, MDI-EB, Methylene diisocyanate–induced eosinophilic bronchitis, MDI-OA, Methylene diisocyanate–induced occupational asthma, MS, Mass spectrometry, NC, Nonatopic healthy control, ROC, Receiver operating characteristic, TDI, Toluene diisocyanate, TDI-OA, Toluene diisocyanate–induced occupational asthma, WRRS, Work-related lower respiratory symptom

Diisocyanates, which are widely used in the manufacture of polyurethane foams, elastomers, adhesives, coatings, insecticides, paints, plastics, and varnishes, may cause occupational asthma as a low-molecular-weight antigen,¹, ² and several efforts have been made to develop serologic markers for isocyanate-induced asthma. In the case of toluene diisocyanate–induced occupational asthma (TDI-OA), specific IgE and IgG antibodies to toluene diisocyanate (TDI)–human serum albumin conjugate were found to be good diagnostic tools.³, ⁴, ⁵ In addition, serum-specific IgG to cytokeratin 19 was suggested as a possible serologic marker of TDI-OA, but the sensitivity of these 2 parameters was too low to be applied.³, ⁴, ⁵, ⁶ In methylene diisocyanate–induced occupational asthma (MDI-OA), although the level of serum specific IgE and IgG antibodies was found to be increased in some patients,⁷ it was not high enough to be used for diagnostic purposes. Considering that permanent impairment of lung function was reported in long-term follow-up studies of isocyanate-induced asthma,⁸ developing relevant serologic markers to identify susceptible subjects among exposed workers is essential.

Proteomic analysis is a complex process involving the purification and identification of individual proteins from all of the proteins expressed in a cell or tissue.⁹ To clarify the pathogenic mechanisms of asthma with proteomic technology, many studies have used human samples such as bronchoalveolar lavage fluid (BALF)¹⁰ and plasma.¹¹, ¹²

In this study, we sought to identify new serologic markers using a proteomic approach and to validate them for screening suspected subjects from among MDI-exposed individuals.

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Methods 

The protocol for this study was reviewed and approved by the Ajou University Institute Review Board, and informed consent was obtained from each study subject.

Study subjects 

Seven subjects with MDI-OA and/or eosinophilic bronchitis (EB), 51 asymptomatic exposed controls (AECs), and 74 nonatopic healthy controls (NCs) were enrolled in this study. Among 7 subjects of the MDI-OA/EB group, 5 had MDI-OA and 2 had MDI-induced EB (MDI-EB). All subjects with MDI-OA/EB and AECs were selected from among 58 previously studied MDI-exposed workers in a single car upholstery factory.⁷ First, 58 subjects were screened by respiratory questionnaire, and 26 subjects who had complained of work-related lower respiratory symptoms (WRRSs) underwent methacholine challenge test. Then, 11 subjects showing a positive response to methacholine challenge test underwent the MDI-specific inhalation tests subsequently, except 2 subjects who refused the MDI-specific inhalation test. Five symptomatic workers who had positive responses to both methacholine challenge test and MDI-specific inhalation challenge were defined as having MDI-OA, and their clinical features are summarized in Table I. WRRS was defined as lower respiratory symptoms aggravated during the work, but improved after the work or during holidays, including cough, sputum, wheezing episodes, or shortness of breath.⁷, ¹³ The subjects with MDI-EB were defined as complaining of WRRS with increased sputum eosinophilia (>3%) after MDI bronchial challenges, but having negative responses to the methacholine bronchial challenge and MDI-specific inhalation challenge tests.¹⁴ Subjects with AEC were defined as subjects who did not have MDI-OA/EB although they had worked in the same workplace. Two subjects who had positive responses to methacholine challenge tests but refused further studies were excluded from the AEC group. The NCs were healthy nonatopic volunteers who had never been exposed to MDI. The detailed results of MDI-specific inhalation challenge were reported in our previous article.⁷

Table I. Clinical features of the study subjects

NA, Not applicable; ND, not done.

Skin prick test and bronchial challenge testing 

The skin prick test, which included 9 common inhalant allergens (tree mixture, grass mixture, mugwort, ragweed, cat fur, dog fur, Dermatophagoides pteronyssinus, Dermatophagoides farinae, and Alternaria; Bencard, Bradford, United Kingdom), was performed on all subjects. The result of each test was reported as the ratio of the mean wheal diameter of the allergen to histamine. For values above 1, the reaction was defined as positive. Atopy was defined as more than 1 positive response to the common inhalant allergens on the skin prick test.

Airway responsiveness to methacholine was tested in those 26 subjects who had WRRS by using the 5-breath dosimeter protocol.¹⁵ MDI-specific inhalation challenge testing was performed on 15 subjects with definite WRRS or positive responses to the methacholine challenge test by using a modified version of a previously described method.⁷, ¹⁶ In brief, 20 mL polymeric MDI (Desmodur 44V20; Bayer, Leverkusen, Germany) was heated to 180°C in a beaker on a hot plate to approach the optimal MDI exposure conditions for bronchial provocation. The atmospheric concentration of MDI was measured serially by using personal samplers (GilAir; Sensidyne Inc, Clearwater, Fla) placed 60 cm away from the hot plate. The initial exposure lasted 20 minutes, and another was performed after a 1-hour break. During the break, the booth was thoroughly ventilated. Spirometric measurements were recorded before the challenge, at 10-minute intervals for 1 hour after the challenge, and subsequently at 1-hour intervals for the next 4 hours. Peak flow measurements were collected at 2-hour intervals for 24 hours. The test was performed in a booth equipped with a ventilating fan and transparent windows through which the subjects were observed. All subjects with MDI-OA had positive results of methacholine challenge, and their PC₂₀ values ranged from 2.76 to 21.93 mg/mL.

Bronchoscopy and bronchoalveolar lavage 

Fiber optic bronchoscopy was performed 24 hours after the MDI-specific challenge test. Bronchoalveolar lavage was performed through a flexible bronchoscope (Olympus BF-P240; Olympus Corp, Tokyo, Japan), which was wedged into the subsegmental bronchus of the right middle lobe. Three 30-mL aliquots of sterile saline solution were instilled through the bronchoscope. The fluid was immediately recovered by gentle suction after each instillation. The harvested BALF was centrifuged at 1000 rpm for 10 minutes, and the supernatant was collected for proteomic analysis according to the MDI-specific inhalation challenge test result. Serum was obtained from each subject after MDI-specific inhalation challenge testing.

Two-dimensional electrophoresis and image analysis 

To evaluate differential protein expression in the MDI-OA and AEC groups, we chose 2 subjects with a more than 25% decrease in FEV₁ after the MDI-specific inhalation challenge test (typical MDI-OA) and 2 subjects with no decrease in FEV₁ after the MDI-specific inhalation challenge test (AECs), and collected BALF for 2-DE.

Each sample was prepared by trichloroacetic acid/acetone precipitation for desalting and concentrating. Immubilized pH gradient (IPG) strips (Immobiline DryStrip, pH 3-10 NL, Amersham Pharmacia Biotech, Uppsala, Sweden) and Pharmalytes (pH 3-10; Amersham Pharmacia Biotech) were used for isoelectric focusing. For the first-dimensional analysis, 1 mg protein was focused on IPG strips as described except that a total of 80,000 voltage-hours was applied.¹⁷, ¹⁸ For second-dimensional separation, electrophoresis was performed by using 9% to 16% gradient polyacrylamide gels until the dye front reached the lower end of the gel. To quantify the relative abundance of the proteins, the gels were stained with Coomassie Brilliant Blue G-250 (Bio-Rad, Hercules, Calif). The stained gels were then scanned using a GS-710 imaging densitometer (Bio-Rad) and analyzed with Image Master Platinum 5 (GE Healthcare, Piscataway, NJ).

In-gel digestion and mass spectrometric analysis 

Protein spots that increased or decreased more than 2-fold between the positive and negative groups were selected for mass spectrometry (MS) analysis. The spots were excised from the gels with a sterile scalpel and placed into Eppendorf tubes. The proteins were then digested by using trypsin (Promega, Madison, Wis) as previously described.¹⁹ For MALDI-TOF/TOF MS, the tryptic peptides were concentrated using a POROS R2, Oligo R3 column (Applied Biosystems, Foster City, Calif). After washing the column consecutively with 70% acetonitrile, 100% acetonitrile, and 0.05 M ammonium bicarbonate, the samples were applied to the R2, R3 column and eluted with cyano-4-hydroxycinamic acid (Sigma, St Louis, Mo). The samples were dissolved in 70% acetonitrile and 2% formic acid before MALDI-TOF/TOF MS.¹⁸ Mass spectra were acquired by using a 4800 Proteomics Analyzer (Applied Biosystems) operated in the MS and MS/MS modes. Peptide fragmentation in the MS/MS mode was achieved by collision-induced dissociation by using atmospheric air as the collision gas. The instrument was operated in reflectron mode and calibrated by using the 4800 calibration mixture (Applied Biosystems); each sample spectrum was further calibrated by using trypsin autolysis peaks. Peptide matching and protein searches were performed by using Mascot and the Swiss-Prot and National Center for Biotechnology Information databases (http://www.matrixscience.com).

Measurement of ferritin and transferrin in sera 

The serum levels of ferritin (Alpha Diagnostic International, San Antonio, Tex) and transferrin (Alpha Diagnostic International) in the subjects from the MDI-OA/EB, AEC, and NC groups were measured by using an ELISA kit according to the manufacturer's instructions.

Statistical analyses 

Log-transformed serum ferritin and transferrin values were compared by ANOVA with Bonferroni correction for multiple testing. All values were adjusted by age and sex by using analysis of covariance (ANCOVA). Receiver operating characteristic (ROC) curves were used to evaluate the validity of the serum ferritin and transferrin levels for discriminating between MDI-OA and AEC, and the area under the curve (AUC) with a 95% CI was computed.²⁰ Sensitivity and specificity were calculated according to the identified optimal cutoffs, and P values were adjusted by age and sex. All computations were performed by using SPSS Version 12.0 (SPSS Inc, Chicago, Ill) and MedCalc (Mariakerke, Belgium).

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Results 

Characteristics of the study subjects 

All subjects were classified into 2 groups, MDI-OA/EB and AEC, on the basis of the results of a methacholine challenge test and MDI-specific inhalation challenge test; their clinical features are summarized in Table I. The NCs were significantly younger than the MDI-exposed workers, including those in the MDI-OA/EB and AEC groups (P < .001), which have been attributable to selection bias. No significant difference was observed in terms of sex, FEV₁(%), or log total IgE among the MDI-OA/EB, AEC, and NC groups, and no difference in work duration was detected among the MDI-exposed subjects.

2-DE analysis of BALF 

To evaluate differential protein expression in the MDI-OA and AEC groups, we chose 2 subjects with a more than 25% decrease in FEV₁ after the MDI-specific inhalation challenge test (typical MDI-OA) and 2 subjects with no decrease in FEV₁ after the MDI-specific inhalation challenge test (AECs), and collected BALF for 2-DE. Five hundred five spots were identified in the gels, of which 28 increased and 89 decreased more than 2-fold on the basis of a comparison of the mean expression level in each group (Fig 1, A and B). Among them, 12 increased and 25 decreased spots were selected for further MS analysis.

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• Fig 1. 

  2-DE separation of BALF proteins from AECs (A) and patients with MDI-OA (B). The proteins identified by MALDI-TOF MS (arrows) are marked by their spot numbers. Arrows indicate the spots corresponding to ferritin (8) and transferrin (1).

Identification of the proteins by MALDI-TOF MS 

Among the 37 protein spots selected for MS analysis, 23 were identified by MALDI-TOF/TOF MS (Table II). Of these, 7 upregulated and 16 downregulated proteins were identified in the subjects with MDI-OA.

Table II. List of proteins identified by MALDI-TOF MS analysis

Cov, Coverage; gi No., genInfo identifier; pI, isoelectric point.

Measurement of the serum ferritin and transferrin levels by ELISA 

To validate the proteins identified by MALDI-TOF/TOF MS, we measured the serum ferritin and transferrin levels in the MDI-OA/EB, AEC, and NC groups (Fig 2), which was adjusted by age and sex. Table III showed both unadjusted and adjusted P values of serum ferritin and transferrin, which indicated that both ferritin and transferrin levels had significant difference between the groups after age and sex adjustment (P = .005 and <.001, respectively).

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• Fig 2. 

  Comparison of the serum concentrations of log ferritin (A) and log transferrin (B) in subjects with MDI-OA/EB, AECs, and NCs. ^∗All data are adjusted by age and sex. Dotted line indicates the mean of each value.

Table III. Comparisons of log ferritin and transferrin values between study groups

Log ferritin	P value	Log transferrin	P value
ANOVA∗	.065	ANOVA∗	<.001
MDI-OA/EB vs AEC	.059	MDI-OA/EB vs AEC	.028
MDI-OA/EB vs NC	.104	MDI-OA/EB vs NC	1.000
AEC vs NC	1.000	AEC vs NC	<.001
ANCOVA†	.007	ANCOVA†	.001
MDI-OA/EB vs AEC	.022	MDI-OA/EB vs AEC	.009
MDI-OA/EB vs NC	.002	MDI-OA/EB vs NC	.458
AEC vs NC	.064	AEC vs NC	.001

Determination of the optimal cutoff levels for serum ferritin and transferrin 

We next evaluated the diagnostic value of serum ferritin (Fig 3, A) and transferrin (Fig 3, B) in discriminating between subjects with MDI-OA/EB and the AECs using the AUC, which was computed from a ROC curve. We then selected the appropriate cutoff values for each ROC curve. Using the cutoff for ferritin (69.84 ng/mL; Table III), the sensitivity and specificity were 85.71% and 71.48%, respectively, with 0.786 (95% CI, 0.614-0.957; P = .053) of the AUC value. When we applied the transferrin cutoff value (≥2.48 μg/mL), the sensitivity and specificity decreased to 71.43% and 51.02%, with 0.612 (95% CI, 0.396-0.829; P = .400) of the AUC value. When both parameters were combined to improve the discriminating power,²¹ the AUC value increased to 0.789 (95% CI, 0.580-0.991; P = .020), whereas the sensitivity and specificity increased to 71.43% and 85.71%, respectively (Table IV).

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• Fig 3. 

  ROC curves for the serum levels of ferritin (A) and transferrin (B), as well as the combined value (C) used to determine the optimal cutoffs for MDI-OA. Arrows indicate the optimal value for ferritin, transferrin, and the combined value (ferritin ≤ 69.84 ng/mL and transferrin ≥ 2.48 μg/mL). Gray regions represent the sensitivity range (60-100) and specificity range (50-100).

Table IV. Sensitivity, specificity, and the values of the area under the ROC with 95% CI and P values

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Discussion 

To elucidate the pathogenic mechanisms of MDI-OA, we adopted a proteomic approach as a screening tool. First, 2-DE was used to screen for target proteins in BALF collected from subjects with MDI-OA and the AECs, and 23 protein spots that were upregulated or downregulated more than 2-fold were selected. Among those spots, transferrin (spot 1), immunoglobin (spot 2), hemoglobin (spots 5 and 6), and vitamin D–binding protein precursor (DBP; spot 7) were upregulated, whereas ferritin light chain (spot 8), gelsolinlike capping protein (spot 12), and multiple annexins (spots 14, 15, and 23) were downregulated.

We found that the ferritin light chain was downregulated whereas transferrin was upregulated in the MDI-OA group compared with the AEC group by using a proteomic approach. Both ferritin and transferrin are regarded as markers of iron metabolism, and they are fully associated with one another. Ferritin is an iron storage protein consisting of 2 subunits, a heavy and light chain, encoded by separate genes that sequester iron in the ferric (Fe³⁺) state. Ferritin is an acute-phase reactant with clinical implications as a serologic marker of acute and chronic inflammation. It often increases during periods of oxidative stress such as acute respiratory distress syndrome (ARDS)²², ²³ or cystic fibrosis²⁴ for the detoxification of free iron, because free iron facilitates the formation of highly toxic hydroxyl radicals from hydrogen peroxide.²⁵, ²⁶ In addition, polymorphisms within the gene that encodes the ferritin light chain are associated with the development of ARDS.²⁷ Thus, altered intracellular and extracellular ferritin expression may have implications in the development of lung inflammation, including ARDS. Consistent with our results, a recent study reported that epithelial cells treated with carbon monoxide (CO) gas, which has a high affinity for ferrous iron and can alter iron metabolism in bronchial epithelial cells,²⁸ exhibited a decreased ferritin concentration. Some studies have shown reduced ferritin levels²⁹ and increased transferrin levels³⁰ in the sera of patients with asthma. Transferrin is an antioxidant that binds iron in the plasma. The iron delivered by transferrin may be associated with increased oxidative cell injury,³¹ because hypotransferrinemic mice are resistant to injury by hyperoxia and metal particles.³², ³³ Although the mechanisms have not been fully clarified, our results indicate that ferritin was downregulated whereas transferrin was upregulated in the BALF of the patients with MDI-OA/EB. These findings were validated by our ELISA results, which showed a significant decrease in serum ferritin in the MDI-OA/EB group compared with the AEC and NC groups, and a higher transferrin level in the MDI-OA/EB group compared with the AEC and NC groups. Serum ferritin and transferrin levels were negatively correlated with one another (r = –0.786; P = .036).

Increased epithelial production of reactive oxygen species is accepted as a major pathogenic mechanism of isocyanate-induced OA. It was previously shown in vitro study that exposure to isocyanates, including TDI, hexamethylene diisocyanate, and MDI, increased the level of intracellular peroxide,³⁴ whereas in vivo antioxidants were found to decrease TDI-induced airway inflammation.³⁵ On the basis of these findings, reactive oxygen species production may be one pathogenic mechanism of MDI-OA. Ferritin and transferrin are both antioxidants, but they have different roles; ferritin is used for detoxification during oxidative stress–induced inflammations, whereas hypotransferrinemia is associated with resistance to oxidative injury. Therefore, some susceptible subjects with defects in iron metabolism and lower serum ferritin levels may develop MDI-OA after MDI exposure, whereas some subjects with lower serum transferrin levels may be resistant to MDI exposure. We speculate that ferritin acts defensively against MDI, and that some subjects with an impaired ferritin level may be more susceptible to MDI-OA than those with a normal iron metabolism when exposed to MDI.

Several upregulated and downregulated proteins were identified in the BALF of the subjects with MDI-OA and the AECs. For example, vitamin DBP was upregulated in the MDI-OA group. Vitamin D metabolites are known to be associated with immune modulation of T_H2 inflammation,³⁶ and genetic polymorphisms of vitamin D receptor are associated with an individual's susceptibility to asthma and atopy.³⁷, ³⁸, ³⁹ Therefore, consistent with our results, vitamin D metabolism seems to contribute to the pathogenesis of asthma. In comparison, gelsolin, a calcium-activated actin filament, was downregulated in the MDI-OA group. Gelsolin has been linked to several pathologic conditions, including inflammation, cancer, and amyloidosis⁴⁰; it is secreted from bronchial epithelial cells and is related to increased mucus production in patients with asthma.⁴¹, ⁴² Finally, multiple annexins were downregulated in the MDI-OA group. Annexins are water-soluble proteins that form voltage-dependent calcium channels within planar lipid bilayers,⁴³ and they are expressed at reduced levels in asthmatic mice.⁴² Additional studies are needed to evaluate the clinical relevance of each protein for MDI-exposed workers.

Developing a useful serologic marker for identifying susceptible patients from among isocyanate-exposed workers is essential. In terms of the long-term prognosis of TDI-OA, it was previously shown that more than 50% of patients with TDI-OA had persistent asthmatic symptoms in spite of complete avoidance.⁸ Therefore, the early detection of susceptible workers is critical to prevent permanent impairment of lung function in patients with isocyanate occupational asthma. Several serologic markers have been considered in an effort to improve the predictability of isocyanate-induced asthma. In the case of TDI-OA, specific IgE and IgG antibodies to TDI–human serum albumin conjugate were suggested as a useful diagnostic marker, but the sensitivity was only around 20% to 30%.³, ⁴⁴ Recently, a volatile form of TDI with 43.9% diagnostic sensitivity was used to discriminate between TDI-OA and AEC.⁵ However, in the case of MDI-OA, only 1 study has shown that serum specific IgG antibodies may be a useful predictive factor (57.1% sensitivity).⁷ In this study, we evaluated the diagnostic value of serum ferritin and transferrin measurements in identifying MDI-OA/EB. We applied ROC curves to compute the optimal values and selected those areas with more than 60% sensitivity and more than 50% specificity (Fig 3). We obtained the optimal AUC values based on those values, although the serum ferritin cutoff value afforded superior sensitivity and specificity compared with the serum transferrin cutoff value (sensitivity, 85.71% vs 71.43%; specificity, 71.48% vs 51.02%). When we combined ferritin with transferrin, however, the P value improved (P value, ferritin only, 0.053; vs ferritin with transferrin, .020). Although a follow-up longitudinal study will be essential to confirm whether these 2 markers will be useful for predicting development of MDI-induced asthma in a larger cohort, on the basis of these findings, we suggest that the 2 combined serologic markers (serum ferritin ≤69.84 ng/mL and serum transferrin ≥2.48 μg/mL) can be used to discriminate between subjects with MDI-OA/EB and the AECs from among MDI-exposed workers with 71.43% sensitivity and 85.71% specificity.

In conclusion, this study is the first to consider differential protein expression in the BALF of subjects with MDI-OA and AECs. We found 23 differentially expressed (more than 2-fold) protein spots after a MDI-specific inhalation challenge test in patients with MDI-OA. Among them, the ferritin light chain was downregulated and whereas transferrin was upregulated, and these results were validated by using sera from exposed workers. Our findings suggest that combined serum ferritin and transferrin (ferritin ≤69.84 ng/mL and transferrin ≥2.48 μg/mL) testing can be used to identify cases of MDI-OA from among the MDI-exposed workers.

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We thank Dr Eun-Young Lee (Yonsei Proteome Research Center, Seoul, Korea) for assistance with the proteomic analysis.

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