Specific serum antibodies against isocyanates: Association with occupational asthma

Specific serum antibodies against isocyanates: Association with occupational asthma Andre Cartier, MD,* Leslie Grammer, MD,** Jean-Luc Malo, MD,* Franqoise Lagier, MD,* Heberto Ghezzo, PhD,* Kathleen Harris, BS,** and Roy Patterson, MD ** Chicago. Ill., and Montreul, Quebec. Canada Although increased levels of specific IgE andlor 1gG antibodies huve been documented in individual cases of occupational asthma caused by common Qpes of isocyanates (hexamethylene diisocyanate (HOI] and diphenylmethane diisocyanate [MDI]), the frequency among workers with occupattonal asthma is still unknown. The sera of 62165 workers referred for specific inhalation challenges with isocyanates were analyzed for the presence of specific antibodies to the relevant tsocyanate. Most workers (39, 63%) were exposed to HDI, some to MDI (17. 27%). and a few to toluene diisocyanate (six. 10%). Specific inhalation challenges were positive in 29 subjects, eliciting either immediate (seven). early late (hvo). late (13), or dual (seven) reactions. Specific inhalation challenges were more often positive in those subjects u>ith increased nonspecific bronchial responsiveness. Tw~enty-nine subjects demonstrated increased levels of specific 1gE andlor IgG antibodies to isocyanates in the absence of antibodies against human serum albumin (increased IgE only, no subject; IgG only, 20; both IgE and IgG. nine subjects). Although there was a loose association between the results of specific inhalation challenges and levels of specific IgE, the association was much better with the level of specifiicIgG. Indeed. 21 of the 29 subjects (72%) with positive challenges had increased levels of specific IgG whereas 25 of the 33 subjects (7670) with negative challenges had normal levels of antibodies. The association was sign&ant with both HDl and MDI. The levels of antibodies were not significantly associated with the type of temporal reaction. We conclude that the levels of specific IgG to the more recent types of isocyanates (HDI and MDI) bear a satisfactory association, in terms of sensitivity and specificity, to the results of specific inhalation chullenges. suggesting mat an immunologic mechanism is im~olved (J ALLERGY CLIN IMMUNOL 1989;84:507-14. )

Occupational asthma is the most common occupational respiratory ailment in the Western world.” Isocyanates are the principal cause. However, despite numerous studies, the pathogenesis of occupational asthma caused by isocyanates is still controversial. In the case of TDI, which was the most frequently used product, specific IgE antibodies have been docu-

From the **Department of Medicine, Sections of AllergyImmunology and Occupational Medicine, NorthwesternUniversity Medical School, Chicago, Ill., andthe *Departmentof Chest Medicine, Hopital du Sac&Coeur, Montreal, Canada. Supported by Ernest S. Hazley Grant and United States Public Health !Iervice Grant AI 11403. Received for publication Nov. 18, 1988. Revised May 30, 1989. Accepted for publication June 7, 19889. Reprint requests:Jean-Luc Malo, MD, Departmentof Chest Medicine, Hopital du Sack-Coeur, 5400 W. Gouin, Montreal, Canada H41 ICS. l/1/14792

Abbreviations used HDI: Hexamethylene diisocyanate HSA: Human serum albumin MDI: Diphenylmethane diisocyanate OD: Optical density PBS: Phosphate-buffered saline PC,,: Concentration of histamine or methacholine (in milligrams per milliliter) causing a 20% fall in FEV, SIC: Specific inhalation challenges TDI: Toluene diisocyanate

mented in <25% of workers in some studies’- ’ and up to 80% by other investigators.6 Other studies failed to detect any specific IgE antibodies at all.‘, ’ Neither could Pagglaro et al.’ detect any specific IgG antibodies. These results may reflect the difficulties in preparing the antigen for antibody assessment or the 507

508 Cattier et al.

J. ALLERGY

fact that this condition is not usually immunologically mediated. Numerous chemically different isocyanates have been introduced in recent years. The most common isocyanatesare HDI and MDI. Specific IgEl’, ” and IgG” antibodies have been detected in individual cases.Specific IgE antibodies appearto be more frequently encounteredwith MD1 than with TDI* Welinder et alI3 recently found an association between exposure to HDI and the levels of specific IgG, but not IgE antibodies. The aim of the present study was to assessthe prevalence of specific IgE and IgG antibodies in 62 workers who were being investigated for possible occupational asthma caused by isocyanates. The diagnosis of occupational asthma was accepted or rejected in every instance through SICs in the laboratory. Most subjects (56, 90%) were exposed to HDI or MDI. MATERIAL Subjects

AND METHODS

All subjects who underwent SICs in our laboratory at Hopital du Sac&-Coeur in Montreal between1984 and 1987 are included in this study with the exception of three subjects in whom the diagnosis remained equivocal after SICs. A total of 62 subjects was thus kept. Six subjects (10%) had been exposed to TDI in the foam industry, 39 (63%) to HDI as spray painters, and 17 subjects (27%) to MD1 in various industries. Of these 17, six subjects were spray painters exposed to polymethylene polyphenylisocyanate, which is composed of approximately 50% of MDI.

Clinical

and functional

investigation

A respiratory and occupational questionnaire was first completed by trained physicians. Spirometry was assessed on a Collins 9 L water spirometer (Warren Collins, Inc., Braintree, Mass.), according to the standards of the American Thoracic Society.14 Skin prick tests were carried out by the prick method with 15 common inhalant allergens. Positive immediate skin reactions were defined as a 23 mm wheal in subjects without dermographism, as assessed by skin testing with PBS, and a 23 mm wheal to histamine phosphate at a concentration of 1 mg/ml. In seven of the 12 subjects who had exhibited increased levels of both specific IgE and IgG antibodies (see Results), skin tests with isocyanates were also performed. Skin prick tests were first done with HSA (5 mg/ml) as a negative control, with histamine phosphate (1 mg/ml) as a positive control, and with TDI-HSA (5 mg/mI), HDI-HSA (5 mg/ml), and MDI-HSA (5 mg/ml). If there was no wheal 10 minutes later, intradermal tests with HSA, TDI-HSA, HDI-HSA, and MDIHSA at a 1: 5000 concentration were performed. In the absence of wheal 10 minutes later, concentrations of 1: 500 and, if this was needed, 1:50 were used. The presence of a wheal was documented 10 minutes, 6, 48, and 72 hours after the introduction of the antigen.

CLIN. IMMUNOL. OCTOBER 1989

Nonspecific bronchial responsiveness to histamine or methacholine was also assessed with a Wright’s nebulizer (output, 0.14 ml/min) to tidal volume breathing for 2 minutes. ”

Specific

inhalation

challenges

These tests were carried out in a challenge room as proposed by Pepys and Hutchcroft.‘” The following sequence of tests was performed on each subject. On the first day of no exposure, spirometry, oral temperature, and white blood count were first assessed. FEV, was monitored every 10 minutes for 1 hour. FEV, and oral temperature were then measured every 30 minutes for 2 hours and hourly for the next 6 hours. Maximum daily fluctuations in FEV, had to be 110% for a subject to continue with the tests. At the end of the day, blood was obtained, and the serum was kept at - 20” C until the antibodies were assessed. On the second day, subjects were exposed to the control product usually mixed with isocyanate, in the case of HDI, an enamel containing aromatic hydrocarbons, ketones, aliphatic and ether ester; in the case of MDI, various aromatic hydrocarbons and fluorocarbons. The diluent used for TDI was a commercial preparation made of polyol (99%) and aliphatic amine (1%). FEV, and oral temperature were monitored as on the first day. On the third day, subjects were exposed to isocyanate in one of the following ways: (1) approximately 100 ml of pure commercial TDI (80% 2,4-TDI and 20% 2,6-TDI) was deposited in a small cup, (2) HDI, which was used for the challenge, was the commercial preparation to which the subject was exposed at work (from 20% to 75% of HDI/HDI biuret, depending of the product), nebulized with the diluent (1: 3 concentration) with an Inspiron nebulizer (Inspiron, Division of C. R. Bard, Inc., Ranch0 Cucamonga, Calif.), and (3) commercial MD1 containing 40% to 50% of diphenylmethane diisocyanate and 50% to 60% of polymethylene polyphenyl isocyanate was heated in a metal cup to approximately 80” C. On the control day, subjects were exposed to the control product for 15 minutes. With the isocyanates, subjects were asked to remain in the challenge room for progressively longer periods of time: one breath, 15 and 45 seconds for a total of 1 minute on the first day, 1 minute, 2 minutes, and 2 minutes for a total of 5 minutes on the second day, and total periods varying from 15 to 120 minutes on the last or second-to-last day. For the final 28 subjects, an MDA7100 monitor (MDA Scientific, Inc., Glenview, Ill.) made possible the constant assessment of levels of isocyanate in the challenge room, which were kept <20 ppb (generally between 5 and 20 ppb). An MDA monitor was not used for the rest of the subjects; however, they did not experience cough, rhinitis, conjunctivitis, nausea, or headaches, all symptoms of toxic reaction to isocyanates. Spirometry and oral temperature were assessed at the same time intervals as on the first and second days. Exposure was stopped if a drop ~20% in FEV, was observed. On the last day, white blood count was reassessed at the end of the day, and in the cases of negative challenges, a histamine or methacholine inhalation test was also repeated. Changes in nonspecific bronchial responsiveness have indeed been found to be

VOLUME NUMBER

Antibodies and asthma to isocvanates

84 4, PAR-r 1

more sensitive thdn spirometry in documenting the effect of exposure to a sensitizing agent.” In the presence of significant changes in PC,,, that is, ?3.2-fold variation.‘” the duration of e-xposure to isocyanate was increased.

Immunologic

tests

These tests were blind to ensure no knowledge of the results of specific inhalation challenges.

Preparation

of chemical

protein

conjugates

The method used to conjugate the various isocyanates to HSA has previously been described.‘” HSA (25% solution) was obtained from the American National Red Cross Blood Services in Washington. D.C. The isocyanate (1 mgimg of HSA) was mixed with the HSA in 7% sodium bicarbonate, stirred at room temperature for 1 hour, dialyzed extensively against PBS, and sterile filtered. As a control, all the above steps were applied to HSA without the addition of an isocyanate. To confirm that conjugation had occurred, the number of free amino groups present in the conjugates was determined according to the method of Snyder and Sobocinski.“’ A’dditionally, immunoelectrophoresis was performed on all the conjugates with Immuno-tee II immunoelectrophoresis plates (Calbiochem-Behring, La Jolla, Calif.) to determine whether conjugation had occurred, as was evidene:ed by altered mobility of the isocyanate-HSA conjugates.

ELISA The ELISA procedure was performed with modifications of methods described elsewhere.2’. ” Briefly, wells of Immulon micro-ELISA plates of polystyrene (Greiner & Sons, Ntirtingen, West Germany) were used. Appropriate antigen coat. serum dilutions, and antibody dilutions had previously been determined with a checkerboard technique. Two hundred microliter volumes were used for all steps, and the plates were washed with PBS-Tween (0.05%, Sigma Chemical Co., St. Louis, MO.) between each layer. The plates were initially coated with isocyanate-conjugate or HSA alone in carbonate buffer, pH 9.6, at a concentration that had been determined to be optimum, and they were incubated overnight at 4” C. All subsequent incubation\ were performed at 37” C. Dilutions of the workers’ sera and the control sera in PBS-Tween were added to the plates and incubated for 1 hour. Rabbit antihuman 1gG or IgE (Calbiochem-Behring) was diluted 1: 1000 in PBS-Tween, added to the wells, and incubated for 45 minutes. Then, alkaline phosphatase-conjugated, goat antirabbit IgG (Sigma Chemical Co.), diluted 1: 1000, was added, and the plates were incubated for 1 hour. Finally. p-mtrophenyl phosphate (Sigma Chemical Co., 104 phosphatase) 1 1 mgiml in diethanolamine buffer, pH 9.8, was added to each well. Known positive control samples were run with all assays, and the assay was stopped when the routine sample reached a previously determined OD. The OD of each well was then read on an Artek automated microELISA reader (Artek Systems Corp., Farmingdale, N.Y.) at 40.5 nm. A worker’s serum was considered to be positive if the OD reading was at least two times the OD of the mean

509

of the negative controls. All assays were performed at least two times and without any activity.

Analysis

of results

Atopy was defined as at least one positive reaction to the common inhalant allergens. Reference values for FEV, were according to Knudson et al.” Dose-response curves to histamine or methacholine were drawn on a noncumulative semilogarithmic scale. PC, value was interpolated from the dose-response curve. Significant bronchial hyperresponsiveness was defined as a PC,, 5 16 mp.!ml.” SlCs were considered positive if the subject had a sustained (at least three consecutive values) fall in FEV, X0% and fluctuations not exceeding 10% on the control day. The type of temporal reaction (immediate. early late. late, or dual) was classitied according to the method of Pcpys and Hutchcroft.‘l Because the presence or absence of occupational asthma had been determined before undergoing specific antibodies assessment in a blind way, the epidemiologic design of the study was of the case-control type.“’ Therefore. odds ratios, instead of sensitivity and specificity used in cohort studies. were assessed for the relationship between the results of specific antibodies and results of SIG. Otherwise. association tables and chi-square analysis were used in the analysis of results. The Fisher’s exact probability test was used for comparisons in which the number was ‘-r.5 in at least one of the cells: otherwise, chi-square was used. Unpaired Student‘s I test was also used to compare the groups of subjects with positive and negative inhalation challenges.

RESULTS SICS Every subject included in this study had symptoms of dyspnea, cough, wheezing, or chest tightness. Ten subjects had at least one symptom, 14 had two, 30 had three, and eight subjects had the four symptoms. Thirty-eight subjects said that their symptoms improved on weekends, and 51 subjects believed their symptoms improved on vacation. Mean duration of exposure to isocyanates was 8.8 t 8.5 years and was not significantly different in subjects with positive challenges (9.2 2 10.2 years) as compared with subjects with negative challenges (8.4 t 6.7 years). As presented in Table I, there was a slight predominance of nonatopic subjects. HDI, MDI, and TDI were the principal types of isocyanates. At the time of the SICs. nearly 25% of the subjects had significant airway obstruction, as determined by a reduced baseline FEV,, with a mean FEV, slighlty lower in the group of subjects with positive challenges. However, significant baseline bronchial obstruction (FEV, 5

80% predicted) was not significantly (chi-square,

more frequent

2.23; p = 0.14) in those subjects with

positive inhalation challenges (9129, 3 1%) than in subjects with a negative challenge (5133, 15%).

510 Cat-tier et al.

TABLE I. Clinical,

J. ALLERGY

functional,

and immunologic

results Positive specific challenges (n = 29)

Sex (M/F) Atopy (present/absent) Smoking habits (smokers/exsmokers/nonsmokers) Duration exposure at work Type of isocyanates TDI MD1 HDI FEV, (% predicted)* Mean Range Number <80% predicted PCzo (mg/ml)t 0.03-0.25 >0.25-2 >2-8 >8-16 >I6 Type of reaction Immediate Early late Late Dual Increased specific antibodies IgE only IgG only Both IgE and IgG HSA

CLIN. IMMUNOL. OCTOBER 1989

Negative specific challenges (n = 33)

All (n = 62)

2613 12/ 17 819112

3211 13120 9/14/10

5814 2.5137 17123122

9.2 + 10.2

8.4 k 6.7

8.8 2 8.5

4 10 15

2 7 24

6 17 39

89 36-124 9

99 68-121 5

94 36-124 14

5 11 6 5 2

0 4 10 5 14

5 15 16 10 16

7 2 13 7

-

-

0 13 8 1

0 7 1 4

0 20 9 5

*FEV, significantly (Student’s I = 2.04; p < 0.05) different in the two groups. ‘/‘Significantly more subjects with a PC, < 16 mg/ml in the group with positive challenges (chi-square, 10.2; p = 0.0014).

Twenty-six percent of subjects did not demonstrate significant bronchial hyperresponsiveness.I9 SICs induced significant bronchoconstriction in nearly half the subjects. Late reactions were the most common. Only three subjects, one who had an early late, one who had a late reaction, and one who had a dual reaction, demonstratedsignificantly high (~37.5” C) temperaturesthe afternoon after the SIC. Sevensubjects had leukocytosis (white blood counts ~10,000). Significant bronchial hyperresponsivenesswaspresent in 27/29 (93%) subjects with positive SICs and in 19/33 (58%) subjectswith negative SICs (chi-square, 10.2; p = 0.0014). Changes in bronchial responsiveness >3.2-fold’* after the challenge occurred in 10 subjects,including sevensubjectswho had positive challengesand three subjectswith negative reactions. In these three subjects, serial monitoring of peak expiratory fldw rates for a minimum of 2 weeks at work disclosedfluctuations suggestiveof work-related asthma. Indices reflecting the concentrations of iso-

cyanates, as assessedin the challenge room in the sevenof the 28 subjectswith positive challenges and in whom such measurementwas carried out, are listed in Table II, demonstratingthat the highest concentrations were <20 ppb. Immunologic

tests

Twenty-nine subjects had increased levels of specific IgE, IgG, or both (Table I). More than half the subjectshad an elevation of specific IgG only. As five other subjects had increased levels of antibodies against HSA and also had increased specific IgE, they were considered as “negative” in the antibody analysis. Skin testing

with

isocyanates

Skin tests were performed on sevenof the 12 subjects who had increased levels of both specific IgE and IgG antibodies. Six subjects were sensitized to HDI, and the other subject was sensitized to MDI.

VOLUME NUMBER

,4ntibodies and asthma to isocyanates

84 4 PAR-- 1

TABLE II. Levels of isocyanates

in subjects

Duration Type of isocyanate

H111 H:)I T’,I

exposure

One breath 6 5

HUI

5 30

TI11 MD1

.-

2 One breath

Type of reaction

lmmediatc Late DU31 Late Dual Late Immediate

Only one subject had a positive immediate skin wheal (5 mm by 5 mm at a concentration of 5 mg/ ml, IO minutes after the introduction of antigen) to HDI-HSA by the prick method. This subject had demonstrated an isolated immediate reaction on exposure to HDI. Two other subjects, one with an isolated immediate reaction and the other subject with a late reaction to HDI, reacted with an induration to the intradermal method at a concentration of 1: 5000 HSA-HDI. Reactions were respectively documented IO minutes (5 mm by 5 mm wheal), and 10 minutes, 6, and 48 hours (5 mm by 5 mm wheal in each instance) after the introduction of the antigen. Association between of antibodies

challenges

of (min)

MD1

with positive

SlCs and levels

There ‘was a loose association between the results of SICs and levels of specific IgE antibodies (Table III). A significant association was found when the levels of specific IgG and the levels of a combination of specific IgG and/or IgE were considered (Table III). The significant association between the results of SICs and levels of specific IgG antibodies was still present by considering only the 28 subjects for whom the monitoring of isocyamates was carried out during the chalbznge procedure. Indeed, six of seven subjects with positive challenges had increased specific IgG levels, and 14 of the 21 subjects with negative chailenges had normal antibody levels (Fisher’s exact test, p = 0.03; odds for increased specific IgG antibodies in a subjects with positive specific inhalation challenge, 88%; odds for normal specific IgG antibodies in a subject with negative inhalation challenge, 67%). The association was highly significant with HDI and MD1 (Table IV). There was no significant association between the type of temporal reactions obtained by SICs ar.d the levels of specific IgE and/or IgG antibodies (Table V). Five of the seven subjects with isolated immediate reactions and 16 of the 22 subjects with nonisolated immediate reactions had elevated IgG levels.

511

Levels (ppb)

__I_

Mean

13.0 13.x I?.? 15.7 I!.7

SD

1 .o -2.3 ; .A.6 7.8 -

Highest

Lowest

5 17 1X 16 10 I8 IL

5 9 12. Y 13 3 II

TABLE III. Association between the results of specific inhalation challenges and of specific antibodies Specific inhalation challenges -___ Positive

Negative

Levels of specific IgE antibodies

Increased* Normalt Levels of specific IgG antibodies IncreasedS Normal3

ii 20

1 32 Fisher’s exact test p = 0.004

8 “I K 25 Chi-square 11.4: p = 0.0001

Levels of specific IgG and/or

IgE antibodies Increased// Normal?

-.,I __ x

8 25

Chi-square 14.3: p = o.ooo1 *Odds for increasedspecific IgE antibodies in a subject with ( + ) specific inhalation challenges. 3 14 tOdds for normal specific IgE antibodies in a subject with ( - ) inhalation challenges, 97%;;. fOdds for increasedspecific IgG antibodies in a subject with t + ) specific inhalation challenges, 72%. POddsfor normal specific IgG antibodies in a suhlect with (-) inhalation challenges, 76%. JjOddsfor increasedspecific IgG and/or IgE antibodiesin a subject with (+ ) specific inhalation challenges, 72%. POddsfor normal specific IgG and/or IgE antibodies in a subject with ( -I inhalation challenges, 76%‘.

DISCUSSION Occupational asthma caused by isocyanates is a common occupational respiratory ailment. ‘. ’ Most of our subjects whose sera were stored between 1984 and 1987 were exposed to the more recently developed

512 Car-tier et al.

TABLE IV. Association antibodies

according

J. ALLERGY

between the results of specific to the type of isocyanates

inhalation

challenges

TDI SIC+

Levels of specific IgE antibodies Increased Normal

0 4

Levels of specific IgG antibodies Increased Normal

SIC+

0 2

1 3

Levels of specific IgE and/or IgG antibodies Increased Normal

and of specific

MDI SIC-

0 2

1 3

0 2

CLIN. IMMUNOL. OCTOBER 1989

HDI SIC-

SIC+

SIC-

2 0 8 7 Fisher’s exact test

7 I 8 23 Fisher’s exact test

p = 0.49

p = 0.003

8 1 2 6 Fisher’s exact test

12 7 3 17 Fisher’s exact test

p = 0.003

p = 0.002

8 1 2 6 Fisher’s exact test

12 7 3 17 Fisher’s exact test

p = 0.003

p = 0.002

ForTDI, the numbersin eachcell aretoo smallto allow comparisons.

TABLE V. Levels of antibodies

according reactions

the type of bronchospastic

Type of temporal Immediate

Levels of specific IgE antibodies Increased Normal Levels of specific IgG antibodies Increased Normal Levels of specific IgE and/or IgG antibodies Increased Normal

Early late

to

reaction late

Dual

0 7

0 2

0 13

0 I

5 2

1 1

9 4

6 1

5 2

1 1

9 4

6 1

isocyanates, HDI and MDI, and worked in secondary industries in which the isocyanate was used but not manufactured. This situation might be representative of what is occurring in several countries. Workers in secondary industries are more likely to be exposed to these new varieties of isocyanates, which are in the form of mono-, oligo-, and polymers. Indeed, although TDI is readily volatile, HDI and MD1 can be detected in the environment once they are sprayed, heated, or involved in a reaction with another product.

As is generally found for low-molecular-weight products, there was a predominance of nonatopic subjects. ‘3’ Every subject, with two exceptions, having positive SICs, demonstrated significant bronchial hyperresponsiveness to histamine. This finding might have been expected, since bronchial hyperresponsiveness can persist after even long intervals away from exposure to the sensitizing agent.‘, * However, some subjects had normal bronchial responsiveness, confirming that a normal PC,, does not refute the diagnosis of occupational asthma, although it does make it less likely. This study demonstrates an association between the presence of antibodies to isocyanates and the results of SICs. To the best of our knowledge, no previous study has related the presence of occupational asthma to the frequency of augmentation in specific IgE and IgG antibodies to these new types of isocyanates, HDI and MDI. The interesting finding was that, although IgG antibodies bore a satisfactory association with the results of the challenges, specific IgE did not. Indeed, 72% of subjects with positive challenges had increased specific IgG antibodies, whereas only 3 1% demonstrated increased specific IgE antibodies. Welinder et a1.13demonstrated an association between the exposure but not the presence of symptoms caused by HDI and the presence of specific IgG but not IgE antibodies directed against the oligomer in 30 car painters. These findings differ from what is generally found with inhalant allergens; indeed, 90% or more

VOLUME NUMBER

Antibodies

84 4. PART 1

of subjects with positive SICs demonstrate increased levels of specific IgE levels to these allergens.‘“. ‘” Increased specific IgG levels to common allergens are encountered less frequently.‘8 The presence of specific 1gG antibodies in 72% of subjects with positive SICs and the absence of such antibodies in 76% of subjects with negative SICs suggest that the mechanism of the reaction is I:;G dependem. However, we acknowledge that the association is not as good as for specific IgE antibodies in the case of common allergens. Failure to detect increased levels of specific IgE or/and IgG antibodies in approximately 30% of our subjects with positive SICs could be due to one or other of the following: First, our challenges could have induced nonspecific reactions. This hypothesis appears unlik’tly. We had the means to control the level of exposure in nearly half of our subjects, and in the other half, symptoms generally associated with toxic reactions t,:) isocyanates (cough, rhinitis. conjunctivitis, nausea. and headaches) were not documented. A relatively small proportion of the reactions (7129, 24%) was of the isolated immediate type. This type of reaction could be difficult to differentiate at times with specific sensitizing immediate reactions. When we analyzed our results only in those 28 subjects for whom molnitoring of isocyanates was carried out during the procedure, the satisfactory association between results of .SL and specific antibodies still hold. Second, if the antibodies were present in amounts below the detect .on limit of the assay, it is possible that the immunologic methods used did not detect all the antibodies. Third, it is possible that the cause of occupational asthma due to isocyanates is multifactorial. Several studies have demonstrated that it is possible to immunize animals against isocyanates. ‘I. 2”.?” Specific IgE and IgG antibodies can then be detected. However, the pharmacologic hypothesis of increased P-adrenergic blockade ori and inhibition of acetylcholinesterase is also possible.3’ ” Although the explanation for the fact that subjects with positive challenges had no detectable specific antibodies is hypothetical? it was predictable that a significant proportion (approximately 25% of subjects in this study) would demonstrate increased levels of specific IgG antibodies at the same time as having negative SICs. Indeed, immunologic sensitization can be detecl.ed before the onset of the disease. It can be arguable that some of our subjects without occupational asthma but with detectable antibodies were not sufficiently exposed to isocyanates with our challenge procedure; indeed, eight of the 33 subjects with negative challenges were exposed to isocyanates for only IS minutes. However, none of the subjects demonstrated significant changes in bronchial responsiveness

and asthma to isocyanates

513

to histamine after this exposure. Such changes are sometimes more sensitive than spirometry in detecting a reaction to a sensitizing agent. ” These findings also demonstrate a different situation to what has been found with TDI. in this instance. specific IgG antibodies were not higher in 9 1 workers with respiratory symptoms caused by TDI compared with control subjects with asthma.” We are not certain of the explanation for the increased levels of antibodies to HSA in some isocyanate workers. It is postulated that anti-HSA antibodies have a normal physiologic function for the removal of aged albumin molecules.” Perhaps isocyanates alter HSA in a manner similar to the usual senescence process, causing the anti-HSA level to be increased and therefore be detectable in higher than usual background levels. We performed skin testing ,iith isocyanate extracts in seven of the 12 subjects with increased specific IgE and IgG antibodies. Positive immediate reaction was documented in one subject, and positive intradermal tests in two other subjects. Skin testing in the evaluation of workers exposed to isocyanates warrants further study since not all of the challenge-positive workers appear to have positive skin test reactions. Baur et al.-’ found positive intradermal reactions in only 26% of symptomatic exposed workers but demonstrated a satisfactory association between the results of RAST and skin testing. The results of this study have practical implications. Indeed, since there is a satisfactory association between the immunologic results and SICs, it might be interesting to use specific IgG assessmentsto monitor the onset of the condition before clinical disease occurs. However. only prospective studies with serial monitoring of IgG antibodies will allow a proper assessment of their role as risk markers for developing occupational asthma. We thank Katherine script.

Tallman

for reviewing

the manu-

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5. Baur X, Dewair M, Fruhmann G. Detection of immunologically sensitized isocyanate workers by RAST and intracutaneous skin tests. J ALLERGYCLINIMMUNOL1984;73:610-8. 6. Patterson R, Hargreave FE, Grammer LC, Hanis KE, Dolovich J. Toluene diisocyanate respiratory reactions. Int Arch Allergy Appl Immunol 1987;84:93-100. 7. Danks JM, Cromwell 0, Buckingham JA, Newman Taylor AJ, Davies RJ. Toluene diisocyanate induced asthma: evaluation of antibodies in the serum of affected workers against a tolyl monoisocyanate protein conjugate. Clin Allergy 198 1; 11: 161-8. 8. Pezzini A, Riviera A, Paggiaro P, et al. Specific IgE antibodies in twenty-eight workers with diisocyanate-induced bronchial asthma. Clin Allergyl984;14:453-61. 9. Paggiaro PL, Filieri M, Loi AM, et al. Absence of IgG antibodies to TDI-HSA in a radioimmunological study. Clin Allergy 1983;13:75-9. 10. Zeiss CR, Kanellakes TM, Bellone JD, Levitz D, Pruzansky JJ, Patteron R. Immunoglobulin E-mediated asthma and hypersensitivity pneumonitis with precipating anti-hapten antibodies due to diphenylmethane diisocyanate (MDI) exposure. J ALLERGYCLIN IMMUNOL1980;65:346-52. 11. Chang KC, Karol MH. Diphenylmethane diisocyanate (MDI)induced asthma: evaluation of the immunologic responses and application of an animal model of isocyanate sensitivity. Clin Allergy 1984;14:329-39. 12. Malo J-L, Ouimet G, Cartier A, Levitz D, Zeiss CR. Combined alveolitis and asthma due to hexamethylene diisocyanate (HDI), with demonstration of crossed respiratory and immunologic reactivities to diphenylmethane diisocyanate (MDI). J ALLERGYCLIN IMMUNOL1983;72:413-9. 13. Welinder H, Nielsen J, Bensryd I, Skerfving S. IgG antibodies against polyisocyanates in car painters. Clin Allergy 1988;18: 85-93. 14. ATS statement. Standardization of spirometry-1987 update. Am Rev Respir Dis 1987;136:1285-98. 15. Cockcroft DW, Killian DN, Mellon JJA, Hargreave FE. Bronchial reactivity fo inhaled histamine: a method and clinical survey. Clin Allergy 1977;7;235-43. 16. Pepys J, Hutchcroft BJ. Bronchial provocation tests in etiologic diagnosis and analysis of asthma. Am Rev Respir Dis 1975;112:829-59. 17. Cattier A, L’Archev&que J. Malo JL. Exposure to a sensitizing occupational agent can cause a long-lasting increase in bronchial responsiveness to histamine in the absence of significant changes in airway caliber. J ALLERGYCLIN IMMUNOL1986;

78:1185-9. 18. Dehaut P, Rachiele A, Martin RR, Malo JL. Histamine doseresponse curves in asthma: reproducibility and sensitivity of different indices to assess reponse. Thorax 1983;38:516-22. 19. Zeiss CR, Patterson R, Pruzansky JJ, Miller MM, Rosenberg

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