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American Journal of Respiratory and Critical Care Medicine Vol 166. pp. 445-450, (2002)
© 2002 American Thoracic Society

Original Article

Diisocyanate Antigen-stimulated Monocyte Chemoattractant Protein-1 Synthesis Has Greater Test Efficiency than Specific Antibodies for Identification of Diisocyanate Asthma

David I. Bernstein, André Cartier, Johanne Côté, Jean-Luc Malo, Louis-Philippe Boulet, Mark Wanner, Joanne Milot, Jocelyne L'Archevéque, Carole Trudeau and Zana Lummus

Department of Internal Medicine, University of Cincinnati, Cincinnati, Ohio; Laboratoire de Pneumologie-Researche, Hôpital du Sacré Coeur de Montreal, Montreal; and Institut de Cardiologie et de Pneumologie de L'Université Laval, Hôpital Laval, Sainte-Foy, Québec, Canada

Correspondence and requests for reprints should be addressed to David I. Bernstein, MD, University of Cincinnati College of Medicine, Department of Internal Medicine, Division of Immunology, 231 Albert Sabin Way, Cincinnati, OH 45267-0563. E-mail: bernstdd{at}email.uc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously reported that diisocyanate-human serum albumin (DIISO-HSA) stimulated production of monocyte chemoattractant protein-1 (MCP-1) by peripheral blood mononuclear cells is significantly associated with a clinical diagnosis of diisocyanate asthma (DA). Others have reported that antibodies for DIISO-HSA are specific but insensitive markers of DA. This study was performed to evaluate test characteristics of the in vitro MCP-1 assay compared with DIISO-HSA–specific immunoglobulin (Ig) G and IgE in identifying workers with DA. MCP-1 was quantitated in peripheral blood mononuclear cell supernatants 48 hours after incubation with DIISO-HSA antigens. Assay results were compared with outcomes of specific inhalation challenge (SIC) testing. Nineteen of 54 (35%) workers assayed for antibodies and MCP-1 stimulation had SIC-confirmed DA. Mean MCP-1 produced by SIC-positive workers was greater than SIC-negative workers (p <= 0.001). Diagnostic sensitivity, specificity, and test efficiency for specific IgG were 47%, 74%, and 65%, respectively, and for specific IgE were 21%, 89%, and 65%, respectively. Sensitivity, specificity, and test efficiency of the MCP-1 test were 79%, 91%, and 87%, respectively. This study indicates that the MCP-1 stimulation assay has greater sensitivity and specificity than the specific antibody assays in correctly identifying DA.

Key Words: occupational asthma • diisocyanate • MCP-1 • antibody


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Approximately 300 substances are known to cause occupational asthma (OA) (1). The diisocyanate class of chemicals is among the leading causes of OA in industrialized countries, affecting 5–10% of chronically exposed workers (2, 3). In industry, the most commonly used agents are 2,4- and 2,6-toluene diisocyanate (TDI), 4,4-diphenylmethane diisocyanate (MDI), 1,5-naphthalene diisocyanate, and 1,6 hexamethylene diisocyanate (HDI). Aromatic diisocyanates, such as TDI and MDI, are used as curing agents and activators for manufacturing surface coatings, varnishes, urethane foams and insulation, adhesives, binders, and sealants. HDI, an aliphatic diisocyanate, and its prepolymers are primarily used as spray paint–hardening agents in automobile body shops (4). Despite implementation of industrial hygiene measures that reduce or eliminate ambient exposure, new cases of OA still occur (5, 6). Therefore, secondary prevention in the form of medical surveillance programs is necessary to identify new cases of diisocyanate asthma (DA) promptly.

Many features of DA resemble allergic asthma. For example, OA is expressed after a latency interval of exposure, and once a worker is sensitized, bronchospasm is elicited by inhalation of subirritant amounts of diisocyanate. Workers with DA have been extensively investigated for evidence of allergic sensitization. Diisocyanates bind readily to a variety of proteins, and in the bronchial lumen, haptenate with autologous lung proteins (especially human serum albumin [HSA]) (7–9). Because specific immunoglobulin (Ig) E antibodies against diisocyanate-modified carrier proteins (e.g., HSA) have been detected in only a subset of workers (30% or less) with confirmed DA (10, 11), alternative immune mechanisms other than immediate hypersensitivity have been investigated (11, 12). We have previously reported that enhanced secretion of monocyte chemoattractant protein-1 (MCP-1) by peripheral blood mononuclear cells (PBMCs) after coincubation with diisocyanate-HSA (DIISO-HSA) is associated with OA (13). This study was performed to assess the diagnostic test characteristics of an in vitro assay of MCP-1 production in comparison to diisocyanate antigen-specific antibody assays for identifying workers with confirmed DA.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects
Fifty-four diisocyanate-exposed workers who participated had prior histories consistent with OA, and were referred for specific inhalation challenge (SIC) testing at two occupational pulmonary disease clinics in Quebec, Canada, located at Laval Hospital in Sainte-Foy, Quebec City (16 workers), and Sacre Coeur Hospital, Montreal (38 workers). Evaluation included methacholine challenge testing using the method of Cockcroft, which was followed by a controlled SIC test to a diisocyanate agent encountered in the workplace (TDI, MDI, or HDI), according to previously described protocols (14, 15). The dose of methacholine required to produce a 20% fall in FEV1 (PC20) was determined by interpolation from the dose response curve. Hyperresponsiveness was defined as a PC20 of 8 mg/ml or less. A decrease in FEV1 of at least 20% from prechallenge baseline during the early and/or late asthmatic response was defined as a positive SIC test. A group of nine nonasthmatic volunteers with no known previous exposure to diisocyanates underwent testing for in vitro MCP-1 production; this reference group was used to define positive and negative in vitro MCP-1 assay responses. Finally, a group of 12 subjects with asthma with no prior diisocyanate exposure and who required daily asthma medications (mean FEV1 = 68% [range, 45–87%] and a mean increase after inhaled albuterol of 28% [range, 13–62%]) were evaluated for in vitro MCP-1 production to diisocyanate antigens; the latter data were compared with diisocyanate-exposed groups who underwent SIC. The institutional review board at each participating institution approved the study, and informed consent was obtained in all subjects.

DIISO-HSA Antigens
HDI-, MDI-, and TDI-conjugated HSA antigens (HDI-HSA, MDI-HSA, TDI-HSA, and mock-conjugated HSA, for use as control HSA) were prepared and characterized as previously described (16), except that HDI molar ratios were determined by mass spectrometry. Conjugates contained 3–10 mol diisocyanate/mol of protein.

Specific Anti-DIISO-HSA Serum Antibodies
Serum-specific IgE and IgG antibodies were assayed in triplicate by indirect enzyme-linked immunosorbent assay (ELISA) (17). Positive reference sera were obtained from TDI-, MDI-, and HDI-sensitized workers. IgE antibody was measured by a double-antibody assay using unlabeled goat anti-human IgE (Kierkegaard and Perry, Gaithersburg, MD), followed by alkaline phosphatase–labeled rabbit anti-goat immunoglobulins. IgG was measured using goat anti-human IgG alkaline phosphatase conjugate (Sigma Chemicals, St. Louis, MO). Tests were read when the positive reference serum (1:10 dilution) triplicates reached a mean optical density (OD405 nm) of 0.6. The antibody response for the DIISO-HSA antigen to which maximal antibody binding (OD) was detected was analyzed. A test was defined as positive for diisocyanate-specific antibody (1) if the mean OD405 nm of test serum (1:10 dilution) triplicates was at least 0.1 and two of more SDs above the mean of eight negative control sera or (2) if the serum tested negative for antibody to control HSA.

Diisocyanate Antigen Stimulation of MCP-1
Blood samples (60–100 ml) were collected in 20-ml siliconized Vacutainer tubes (Becton Dickenson, Franklin Lakes, NJ) containing 2.8 ml of citrate phosphate dextrose (CPD) anticoagulant (161.5 mM D-glucose, 88.4 mM sodium citrate · 2 H2O, 15.5 mM citric acid, 16.1 mM NaH2 PO4 · H2O) and shipped by overnight carrier to the University of Cincinnati Allergy Laboratory (Cincinnati, OH). Mononuclear cells were purified by isopycnic density gradient centrifugation on endotoxin-tested Histopaque-1,077 (Sigma Chemicals, St. Louis, MO), washed twice with Hank's balanced salt solution containing 0.01 M ethylenediaminetetraacetic acid, and resuspended in RPMI 1640, 10% heat-inactivated fetal bovine serum, 25 mM HEPES, sodium pyruvate, and penicillin streptomycin. Purified PBMCs were 95–99% viable and consisted of 81–89% lymphocytes, 9–14% monocytes, 2–5% granulocytes, less than 0.7% eosinophils, and less than 0.2% basophils. One milliliter of 5 x 106/ml mononuclear cells was added to 2-ml 24-well tissue culture plates and cultured for 48 hours in 37°C 5% CO2 in medium alone or in 50 µg/ml of the following stimulator: phytohemagglutinin A, mock-conjugated HSA, TDI-HSA, MDI-HSA, or HDI-HSA. Supernatants were stored at -80°C until assayed.

MCP-1 was quantitated in PBMC culture supernatants with a commercial immunoassay (Endogen, Woburn, MA). MCP-1 stimulation was derived by subtraction of MCP-1 amounts measured in cell cultures incubated in medium alone from that measured in supernatants of antigen-stimulated PBMCs. Medium control background values subtracted (in mean ng/ml ± SD) for MCP-1 were 54 ± 54 for SIC-positive workers, 99 ± 148 for SIC-negative workers, and 6 ± 4 for nonexposed control subjects. The raw data were expressed as maximal antigen stimulation or the highest amount of MCP-1 stimulation measured in any of the DIISO-HSA cultures. A test was positive if the maximal antigen stimulation of MCP-1 was two or more SDs above the mean MCP-1 amount quantitated in PBMC culture supernatants of a group (n = 9) of nonasthmatic volunteers with no diisocyanate exposure. Laboratory personnel performed assays before SIC tests were performed and were subsequently blinded to the final results of the SIC tests.

Data Analysis
For determination of test characteristics, antibody and MCP-1 results were treated as categorical data and were classified as true positives, false negatives, true negatives, or false positives by comparison to results of SIC, which was considered the gold standard for confirming a diagnosis. Test characteristics were calculated as follows: sensitivity = (true positive/[true positive + false negative]) x 100; specificity = (true negative/[true negative + false positive]) x 100; predictive value of a negative test = (true negative/[true negative + false negative]) x 100; predictive value of a positive test = (true positive/[true positive + false positive]) x 100; and test efficiency (percentage of subjects correctly classified) = ([true positive + true negative]/[true positive + false positive + true negative + false negative]) x 100. Test characteristics were also examined in workers recently exposed at work to diisocyanates (within the previous 6 months) and in those who had last been remotely exposed more than 6 months before the evaluation. Because the Shapiro Wilks test showed a significant departure from normality for each outcome variable (p < 0.01), relationships between quantitative data (i.e., MCP-1 and PC20 or specific antibody) were evaluated by categorical analyses (chi-square test or Spearman rank-order test).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Study Population
Of 54 diisocyanate-exposed workers evaluated, 8 (15%) had prior work exposure to TDI, 36 (67%) to HDI, and 10 (18%) to MDI. The worker population consisted of 52 males and 2 females with a mean age of 39.9 years. The mean period of active diisocyanate exposure was 134.8 ± 133.1 months. At the time of evaluation, 39 workers had recent or current exposure to isocyanates (last exposure within the previous 6 months; mean time since last exposure was 1.0 [range, 0–5 months]), and 15 workers had been remotely exposed (last exposure was more than 6 months before studies; the mean time since the last exposure was 22.1 [range, 7–96 months]). Based on results of the SIC, DA was confirmed in 19 of 54 workers (35%) and was excluded in 35 workers.

Bronchoprovocation Studies
Methacholine testing was performed in 54 workers before the initiation of SIC. The geometric mean PC20 was 2.31 mg/ml in the SIC-positive workers (Group 1) and 8.27 mg/ml in the SIC-negative workers (Group 2). In 52 workers in whom PC20 was measured before SIC, 12 of 18 (66%) Group 1 workers had airway hyperresponsiveness (PC20 of 8 mg/ml or less) compared with 15 of 34 (44%) Group 2 workers. Of 19 workers with positive SIC tests, four had early asthmatic responses (mean percentage decrease in FEV1 of 29.3 ± 14.0), nine had isolated late-phase responses (mean percentage decrease in FEV1 of 28.3 ± 6.7), and six had dual responses (mean percentage decrease in FEV1 of 28.2 ± 10.7 during the early response, followed by mean percentage decrease in FEV1 of 27.9 ± 6.5 during the late response).

Specific DIISO-HSA Antibody Studies
The diagnostic test characteristics of antibody studies are shown in Table 1. Positive or negative antibody status was determined in the ELISA test for the DIISO-HSA antigen that yielded maximal antibody binding measured as OD. Serum-specific IgG and specific IgE were detected in 9 of 19 (47% sensitivity) and 4 of 19 (21% sensitivity) workers with positive SIC responses, respectively. Because of a higher false-positive rate in IgG antibody producers, specificity of specific IgG was 74% compared with 89% for the specific IgE antibody. Significant binding (OD two or more SD above the mean of control sera) to mock-conjugated HSA was not detected in any worker with specific IgE or IgG binding to DIISO-HSA antigen.


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  		TABLE 1. Characteristics of diiso-hsa–specific igg and ige antibody tests in diisocyanate-exposed workers based on results of specific inhalation challenge testing

 
Specific IgE was detected in some workers with OA caused by HDI (2 of 12) or MDI (2 of 2) but was not found in workers with confirmed TDI asthma (zero of five). DIISO-HSA serum-specific IgG or IgE was not significantly associated with the presence of either a late phase or early asthmatic response during SIC (Fisher's test).

Specific IgG was detected in 5 of 8 (63%) SIC-positive workers with remote exposure to diisocyanates compared with 4 of 11 (36%) with recent exposure. Despite the greater sensitivity of specific IgG in identifying remotely exposed workers with DA, specificity was less than for recently exposed workers (57% versus 79%). The overall predictive values (both positive and negative) and test efficiencies for specific IgG and IgE immunoassays ranged between 40% and 76% (Table 1).

In vitro Assay of MCP-1 Enhancement
As shown in Figure 1 , diisocyanate antigen stimulation of MCP-1 synthesis was significantly increased in the SIC-positive group of workers compared with the challenge-negative workers or the control group of nonexposed subjects. The SIC-positive group showed higher MCP-1 production than challenge-negative workers by PBMCs stimulated with each of the three individual diisocyanates (p <= 0.01). Analysis of the data for MCP-1 stimulation of PBMCs cultured with the diisocyanate antigen to which the worker was exposed (workplace relevant antigen shown in Figure 1) also showed a significant increase in SIC-positive workers compared with SIC-negative workers (p < 0.001). The difference between SIC-positive and SIC-negative workers was especially marked when the data were analyzed for the maximal response to any of three diisocyanates tested (p < 0.001).



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  		Figure 1. Mean values of MCP-1 stimulation in two groups of workers undergoing SIC testing, including SIC-positive (n = 19) and SIC-negative (n = 35), as well as a group of nine nonexposed, nonasthmatic control subjects. MCP-1 stimulation is MCP-1 measured in culture supernatants of antigen-stimulated PBMCs at 48 hours minus MCP-1 in supernatant of cells cultured in medium alone. Responses are shown to individual diisocyanate antigens (MDI-HSA, TDI-HSA, HDI-HSA), control subjects (PHA, HSA), diisocyanate antigens eliciting maximum stimulation (Max), and the work-relevant DIISO-HSA antigen. Significant differences between SIC-positive and SIC-negative groups are indicated as p values (Mann-Whitney). Error bars show SEM.

 
The data for maximal MCP-1 stimulation are shown in Figure 2 . In recently exposed workers (Figure 2A), all but three of the SIC-positive workers exhibited positive MCP-1 production as defined by MCP-1 of two SD or more above the mean of the control group; there were three workers in the SIC-negative group with false-positive results. Based on maximal MCP-1 stimulation, remotely exposed workers with DA could be discriminated from SIC-negative workers with the exception of one false-negative result (Figure 2B).



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  		Figure 2. Mean MCP-1 stimulation (antigen-stimulated minus medium control) in supernatants of cultured PBMCs from SIC-positive and SIC-negative workers, showing maximal stimulation in recently exposed workers (A) and in remotely exposed workers who had been removed from diisocyanate exposure for at least 6 months (B). The positive threshold (dashed line) for a positive MCP-1 result was determined as two standard deviations above the mean of the nonexposed control group.

 
The overall sensitivity and specificity of the MCP-1 assay was 79% and 100%, respectively (Table 2). The test performed best in remotely exposed workers or those whose last diisocyanate exposure was at least 6 months before SIC. In this group, specificity was 100%. Sensitivity was 88%, and the predictive value of a positive test was 100% for identifying DA.


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  		TABLE 2. Test characteristics of antigen-stimulated MCP-1 in diisocyanate-exposed workers based on results of specific inhalation challenge testing

 
As mentioned, airway hyperresponsiveness (PC20 of 8 mg/ml or less) was detected in 66% of Group 1 (SIC-positive workers) compared with 44% of Group 2 (SIC-negative workers). The MCP-1 amounts were analyzed separately in subgroups that exhibited increased (PC20 of 8 mg/ml or less) and normal (PC20 = 8 mg/ml) methacholine responsiveness. As shown in Figure 3 , MCP-1 amounts were significantly elevated in SIC-positive workers when compared with SIC-negative workers, regardless of whether workers were responsive (p <= 0.05) or nonresponsive to methacholine (p <= 0.005). Categorical analysis found no significant association between MCP-1 and methacholine PC20 (chi square, p = 0.18) in 52 workers for whom both data sets were available, even after correcting for IgG (p = 0.73) and IgE antibody status (p = 0.67). The MCP-1 assay was also tested in a control group of 12 subjects with asthma without previous diisocyanate exposure (Figure 4) . There were no significant differences in maximal MCP-1 responses between SIC-negative and the control subjects with asthma; maximal MCP-1 responses in SIC-positive diisocyanate workers were significantly greater than SIC negative (p = 0.003) and the nonexposed groups with asthma (p = 0.026).



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  		Figure 3. MCP-1 stimulation (antigen-stimulated minus medium control) in supernatants of cultured PBMCs from challenge-positive (SIC-positive) and challenge-negative (SIC-negative) workers, showing maximal stimulation in hyperresponsive workers, defined as baseline prechallenge PC20 of 8 mg/ml or less methacholine (A) and in nonhyperresponsive workers (PC20 of more than 8 mg/ml methacholine) (B). The positive threshold (dashed line) for a positive MCP-1 result was defined as two standard deviations above the mean of the nonexposed, nonasthmatic reference group.

 


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  		Figure 4. In vitro MCP-1 stimulation of SIC-positive workers (n = 19) was compared with SIC-negative workers (n = 35, p=0.003) and with asthmatic subjects (n = 12, p=0.026) with no current or prior diisocyanate exposure (Mann-Whitney). The positive threshold (dashed line) for a positive MCP-1 result was determined as two standard deviations above the mean of the nonexposed, nonasthmatic reference group.

 
TDI workers represented a small proportion (n = 8) of the total population relative to workers with prior exposure to HDI or MDI (n = 46). Although sensitivity of MCP-1 was lower in TDI workers (60% versus 86% for HDI or MDI), specificity of TDI-induced MCP-1 was 100% versus 81% in the HDI/MDI-exposed group.

The Spearman rank order correlation method was used to test for a relationship between the maximum amounts of serum specific antibody reactive with any diisocyanate conjugate tested (estimated from ELISA OD units relative to an OD of 0.6 for positive reference sera) and quantitative amounts of MCP-1 production in vitro by antigen stimulated PBMCs. Correlation coefficients were 0.21 for MCP-1/IgE (p = 0.137) and 0.25 for MCP-1/IgG (p = 0.07), indicating that there was no significant association between quantities measured for MCP-1 and either antibody isotype.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A simple diagnostic test able to discriminate DA from non-OA would be most useful. Achieving this goal has been historically problematic for OA caused by chemicals, as the diagnosis is often difficult to confirm, requiring either controlled SIC testing or supervised workplace monitoring of lung function (18, 19). Results obtained in this study on patients whose diagnoses of DA were either rigorously confirmed or excluded with SIC testing indicate that the in vitro maximal MCP-1 stimulation assay is more sensitive and more predictive than diisocyanate antigen-specific IgG or IgE assays in identifying workers with DA.

As shown in Figure 1, maximal diisocyanate-stimulated MCP-1 amounts were significantly increased in SIC-positive versus SIC-negative workers. It is noteworthy that the composition of our study population was heterogenous with regard to current asthma status defined by responsiveness to methacholine. In fact, asthma severity (as defined by mean PC20) was four-fold greater in SIC-positive (Group 1) versus SIC-negative workers (Group 2). To determine whether elevated MCP-1 was associated with the presence or absence of asthma rather than specific bronchial sensitivity to diisocyanates, we analyzed subgroups of workers with and without positive methacholine tests. As shown in Figure 3, MCP-1 was still significantly elevated in SIC-positive workers regardless of whether subjects exhibited responsiveness to methacholine. Furthermore, a categorical analysis was performed in Group 1 and Group 2 workers to determine whether MCP-1 amounts correlated with nonspecific airway hyperresponsiveness. This analysis failed to demonstrate any significant associations between PC20 and MCP-1 amounts even after correcting for other independent variables, including antibody status. In addition, maximal stimulation amounts of MCP-1 among SIC challenge-positive workers were significantly increased above what was assayed in a group of nonexposed subjects with clinically active asthma (Figure 4) with only one of the latter subjects exhibiting a positive MCP-1 response. Thus, the absence of significant relationships between maximal MCP-1 production and current asthma status defined by either reversible airway obstruction or methacholine responsiveness supports the validity of the MCP-1 assay as a specific diagnostic marker of DA.

At the same time, the MCP-1 assay exhibited equivalent specificity to ELISA antibody tests. There was also a trend suggesting greater sensitivity in remotely exposed versus recently exposed workers. Specificity was very high in the subgroup of TDI-exposed workers (100%), but sensitivity was less than that observed among the combined group of HDI-MDI–exposed workers (60% versus 86%). Because of the small number of TDI workers (n = 8), no conclusions can be inferred with regard to these observations.

In the study population, there was marked heterogeneity in terms of the type and pattern of prior diisocyanate exposure. The cumulative exposure and exact composition (e.g., monomeric versus prepolymeric) of diisocyanate chemicals encountered by each worker were unknown. However, differences between workers in time duration since last workplace exposure could be addressed by stratification into recently and remotely exposed cohorts.

Our previous studies indicated that it was not possible to predict in any particular subject which DIISO-HSA antigen would be most efficient in stimulating MCP-1 (13). Therefore, MCP-1 responses to a panel of three diisocyanate antigens were determined, and the antigen eliciting the maximal MCP-1 stimulatory response was analyzed. In fact, mean MCP-1 stimulation with single chemical antigens (TDI-, MDI-, or HDI-HSA) and with the work relevant DIISO-HSA antigen (prepared with the diisocyanate encountered at work) was less optimal than was maximal MCP-1 stimulation in differentiating SIC-positive from SIC-negative workers (Figure 1). Nevertheless, the MCP-1 response is believed to have specificity for diisocyanate antigenic determinants, as no response has previously been observed to a structurally unrelated chemical conjugate, phthalic anhydride-HSA (13).

DIISO-HSA antigens exhibit antigenic crossreactivity in serologic studies and an antibody-positive serum is capable of binding to multiple diisocyanate antigens. As is generally true for hapten-protein antigens, patient sera may contain a heterogeneous mixture of antibodies, some of which react with both the homologous diisocyanate and also crossreact with heterologous diisocyanates (20, 21) and others that are specific exclusively for the homologous diisocyanate (22, 23). Crossreactions occurring in cellular responses to diisocyanate derivatives have also been demonstrated (24). Crossreactions between aromatic (MDI and TDI) and aliphatic (HDI) compounds have been attributed to new and similar epitopes formed in the carrier protein during the chemical reaction with the diisocyanate group (4, 24).

In agreement with previous studies, specific IgE was insensitive as a diagnostic indicator of DA (21% sensitivity) but possessed reasonably high overall diagnostic specificity (89%) (10, 25). Our results confirm the test characteristics of specific IgE reported by Cartier and colleagues using a similar ELISA method (sensitivity of 31% and specificity of 97%), even though the criterion for defining a positive response differed from our study (10). It should be emphasized that in the latter study, elevated specific IgE was detectable in 7 of 15 (47%) workers with HDI-induced asthma, 2 of 10 (20%) with MDI asthma, and was absent in 4 workers with TDI asthma. It is a curious phenomenon that workers exposed to TDI differ from other diisocyanate workers in that they rarely produce serum-specific IgE antibodies, although IgG antibodies may be produced. In this study, 4 of 16 (25%) and 2 of 2 workers with HDI and MDI asthma, respectively, exhibited specific IgE; 0 of 5 workers with TDI asthma were specific IgE positive. The combined sensitivity of specific IgE for HDI and MDI asthma was 26% in this study compared with 36% reported in the study of Cartier and colleagues. Therefore, it is clear that diisocyanate antigen serum-specific IgE may be meaningful, if positive, in HDI and MDI but not in TDI-exposed workers but lacks the overall sensitivity needed for medical screening.

Test characteristics of serum-specific IgG were also evaluated. Cartier and colleagues reported that specific IgG assays exhibit 72% and 76% sensitivity and specificity, respectively (10), versus 47% and 74% in our study. Park and associates recently reported that TDI-HSA–specific IgG measured by ELISA had 46% sensitivity and 92% specificity in identifying TDI asthma confirmed by SIC (26). The role of specific IgG in DA, whether pathogenic, beneficial, or irrelevant, is unknown. In a group of nine asymptomatic MDI-exposed foam workers who were negative for antibodies to HSA, we detected MDI-specific IgE in two (22%) workers and MDI-specific IgG in one of the IgE-positive workers (12). IgG antibodies reactive with MDI-HSA were also found in two other groups of asymptomatic MDI-exposed workers (27, 28). Of the 25 HDI-exposed asymptomatic subjects studied in this article, 5 (20%) produced diisocyanate-specific IgG antibodies, and 3 (12%) produced IgE specific antibodies (data not shown). Another group has reported a somewhat higher prevalence of HDI-HSA IgG antibody responses in 34% of asymptomatic HDI-exposed auto body shop workers (29).

Intuitively, if IgG were merely an index of exposure, one would expect to find a higher prevalence of specific antibody responses among workers with recent (6 months or less) rather than remote exposure. In the population under study, we found the reverse to be true. Specific IgG was detected in 10 of 39 (26%) of all recently exposed workers, versus 8 of 15 (53%) of remotely exposed workers. In the confirmed DA workers, IgG was found in 4 of 11 (36%) of recently exposed and 5 of 8 (63%) remotely exposed. A similar trend was observed in the MCP-1 assay, in which 73% of recently exposed DA workers and 88% of remotely exposed DA workers had positive tests for MCP-1. The increased sensitivity for both antibody and MCP-1 in remotely exposed workers cannot be explained by differences in chemical exposure as there was no correlation found between duration of exposure of DA patients and production of IgG, IgE, or MCP-1. The diminished signals observed in both immune assays among recently exposed subjects suggest that immune responses in these workers are downregulated. Exposure of human subjects to allergenic chemicals by the intravenous or mucosal routes can result in specific immunologic unresponsiveness (30). It is therefore possible that responses to diisocyanates in these workers are influenced by immunologic suppression or specific immunologic tolerance, which wanes with time after cessation of exposure. This hypothesis deserves further investigation.

In summary, specific IgE possessed reasonably high specificity for identifying challenge positive workers. Despite similarity of these results with other laboratories, it is difficult to make interlaboratory comparisons of antibody data, when there are notable differences in antigen conjugates, use of control sera, and criteria used for defining a positive antibody result. This emphasizes the need to develop standard protocols, antigens, and control sera, which could be shared between laboratories. The absence of antibodies in the vast majority of workers with proven DA indicates that antibody formation does not have a causal relationship to DA. Therefore, alternative immunologic or nonimmunologic mechanisms exist for the pathogenesis of DA. These findings indicating a strong association between diisocyanate antigen enhancement of MCP-1 and DA suggest that further investigation and validation of cellular immunoassays could enable development of more sensitive and specific diagnostic tests that could be useful in the diagnosis of OA.


   Acknowledgments
 
:

The authors wish to thank Linda Levin, Ph.D. (Center for Biostatistical Services, University of Cincinnati), for statistical assistance; Greg Agnackzek, Ph.D. (Biophysics Department, University of Cincinnati), for performance of mass spectrometry analysis of HDI antigens; and Jocelyn Biagini for technical assistance.

This study was supported by the International Isocyanate Institute and by research grant RO1 OH03519-01 from the National Institute for Occupational Safety and Health, Centers for Disease Control.

Received in original form September 6, 2001; accepted in final form March 6, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bernstein DI, Bernstein IL. Occupational asthma. In: Middleton E, Reed CE, Ellis EF, Adkinson NF, Yunginger JW, Busse WW, editors. Allergy principles and practice, 5th ed. St. Louis, MO: Mosby Yearbook; 1998. p. 963–980.
  2. Meredith SK, Taylor VM, McDonald JC. Occupational respiratory disease in the UK: a report to the British Thoracic Society and the Society of Occupational Medicine by the SWORD project group. Br J Ind Med 1989;48:292–298.
  3. Tarlo SM, Liss GM, Dias C, Banks DE. Assessment of the relationship between isocyanate exposure levels and occupational asthma. Am J Ind Med 1997;32:517–521.[CrossRef][Medline]
  4. Bernstein IL. Isocyanate-induced pulmonary diseases: a current perspective. J Allergy Clin Immunol 1982;7:24–31.
  5. Bernstein DI, Korbee L, Stauder T, Bernstein JA, Scinto J, Herd ZL, Bernstein IL. The low prevalence of occupational asthma and antibody-dependent sensitization to diphenylmethane diisocyanate in a plant engineered for minimal exposure to diisocyanates. J Allergy Clin Immunol 1993;92:387–396.[CrossRef][Medline]
  6. Kraw M, Tarlo SM. Isocyanate medical surveillance: respiratory referrals from a foam manufacturing plant over a five-year period. Am J Ind Med 1999;35:87–91.[CrossRef][Medline]
  7. Lange RW, Day BW, Lemus R, Tyurin VA, Kagan VE, Karol MH. Intracellular S-glutathionyl adducts in murine lung and human bronchoepithelial cells after exposure to diisocyanatotoluene. Chem Res Toxicol 1999;12:931–936.[CrossRef][Medline]
  8. Wisnewski AV, Srivastava R, Herick C, Xu L, Lemus R, Cain H, Magoski NM, Karol MH, Bottomly K, Redlich CA. Identification of human lung and skin proteins conjugated with hexamethylene diisocyanate in vitro and in vivo. Am J Respir Crit Care Med 2000;162:2330–2336.[Abstract/Free Full Text]
  9. Redlich CA, Karol MH, Graham C, Homer RJ, Holm CT, Wirth JA, Cullen MR. Airway isocyanate-adducts in asthma induced by exposure to hexamethylene diisocyanate. Scand J Work Environ Health 1997;23:227–231.[Medline]
  10. Cartier A, Grammer L, Malo JL, Lagier F, Ghezzo H, Harris K, Patterson R. Specific serum antibodies against isocyanates: association with occupational asthma. J Allergy Clin Immunol 1989;84:507–514.[CrossRef][Medline]
  11. Gallagher JS, Tse CST, Brooks SM, Bernstein IL. Diverse profiles of immunoreactivity in toluene diisocyanate asthma. J Occup Med 1981;23:610–616.[CrossRef][Medline]
  12. Herd ZL, Bernstein DI. Antigen-specific stimulation of histamine releasing factors in diisocyanate-induced occupational asthma. Am J Respir Crit Care Med 1994;150:988–994.[Abstract]
  13. Lummus ZL, Alam R, Bernstein JA, Bernstein DI. Diisocyanate antigen-enhanced production of monocyte chemoattractant protein-1, IL-8, and tumor necrosis factor-{alpha} by peripheral mononuclear cells of workers with occupational asthma. J Allergy Clin Immunol 1998;102:265–274.[CrossRef][Medline]
  14. Ryan G, Dolovich MB, Obminski G, Cockcroft DW, Juniper E, Hargreave FE, Newhouse M. Standardization of inhalation provocation tests: influence of nebulizer output, particle size, and method of inhalation. J Allergy Clin Immunol 1981;67:156–161.[CrossRef][Medline]
  15. Sterk PJ, Fabbri LM, Quanjer PH, Cockcroft DW, O'Byrne PM, Anderson SD, Juniper EF, Malo J-L. Airway responsiveness: standardized challenge testing with pharmacologic, physical, and sensitizing stimuli in adults. Eur Respir J 1993;5:53–83.
  16. Lummus ZL, Alam R, Bernstein JA, Bernstein DI. Characterization of histamine releasing factors in diisocyanate-induced occupational asthma. Toxicology 1996;111:191–206.[CrossRef][Medline]
  17. Hornbeck P. Enzyme-linked immunosorbent assays. In: Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W, editors. Current protocols in immunology. New York: Greene Publishing and Wiley-Interscience; 1991. p. 2.1.6–2.2.6.
  18. Malo JL, Ghezzo H, L'Archeveque J, Lagier F, Perrin B, Cartier A. Is the clinical history a satisfactory means of diagnosing occupational asthma? Am Rev Respir Dis 1991;143:528–532.[Medline]
  19. O'Brien IM, Newman-Taylor AJ, Burge PS, Harries MG, Fawcett IW, Pepys J. Toluene diisocyanate induced asthma. II. Inhalation challenge tests and bronchial reactivity studies. Clin Allergy 1979;9:7–15.[CrossRef][Medline]
  20. Baur X. Immunologic cross-reactivity between different albumin-bound isocyanates. J Allergy Clin Immunol 1983;71:197–205.[CrossRef][Medline]
  21. Butcher BT, O'Neil CE, Reed MA, Salvaggio JE. Radioallergosorbent testing with p-tolyl monoisocyanate in toluene diisocyanate workers. Clin Allergy 1983;13:31–34.[CrossRef][Medline]
  22. Grammer LC, Harris KE, Malo J-L, Cartier A, Patterson R. The use of an immunoassay index for antibodies against human protein conjugates and application to human isocyanate disease. J Allergy Clin Immunol 1990;86:94–98.[CrossRef][Medline]
  23. Aul D, Bhaumik A, Kennedy AL, Brown WE, Lesage J, Malo J-L. Specific IgG response to monomeric and polymeric diphenylmethane diisocyanate conjugates in subjects with respiratory reactions to isocyanates. J Allergy Clin Immunol 1999;103:749–755.[CrossRef][Medline]
  24. Baur X, Seeman U, Marczynski B, Chen Z, Raulf-Heimsoth M. Humoral and cellular immune responses in asthmatic isocyanate workers: report of two cases. Am J Ind Med 1996;29:467–473.[CrossRef][Medline]
  25. Tee RD, Cullinan P, Welch J, Burge PS, Newman-Taylor AJ. Specific IgE to isocyanates: a useful diagnostic role in occupational asthma. J Allergy Clin Immunol 1998;101:709–715.[CrossRef][Medline]
  26. Park HS, Kim HY, Nahm DH, Son JW, Kim YY. Specific IgG, but not specific IgE, antibodies to toluene diisocyanate-human serum albumin conjugate are associated with toluene diisocyanate bronchoprovocation test results. J Allergy Clin Immunol 1999;104:847–851.[CrossRef][Medline]
  27. Liss GM, Bernstein DI, Moller DR, Gallagher JS, Stephenson RL, Bernstein IL. Pulmonary and immunologic evaluation of foundry workers exposed to methylene diphenyldiisocyanate (MDI). J Allergy Clin Immunol 1988;82:55–61.[CrossRef][Medline]
  28. Lushniak BD, Reh CM, Bernstein DI, Gallagher JS. Indirect assessment of 4,4'-diphenylmethane diisocyanate (MDI) exposure by evaluation of specific humoral immune responses to MDI conjugated to human serum albumin. Am J Ind Med 1998;33:471–477.[CrossRef][Medline]
  29. Redlich CA, Stowe MH, Wisnewski AV, Eisen EA, Karol MH, Lemus R, Holm CT, Chung JS, Sparer J, Liu Y, et al. Subclinical immunologic and physiologic responses in hexamethylene diisocyanate-exposed auto body shop workers. Am J Ind Med 2001;39:587–597.[CrossRef][Medline]
  30. Chase M. The induction of tolerance to allergenic chemicals. Ann NY Acad Sci 1982;392:228–247.[Medline]



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