VOLUME NUMBER
23.
24. 25.
26.
27.
87 3
Reactions
Late, but not early, asthmatic reactions induced by toluene diisocyanate are associated with increased airway responsiveness to methacholine. Em .I Respir Dis 1986;69:276-84. Block G, Tse KS, Kijek K, Chart-Yeung M. Baker’s asthma. Clinical and immunological studies. Clin Allergy 1983;13:35970. Cartier A, Malo JL, Dolovich J. Occupational asthma in nurses handling psyllium. Clin Allergy 1987;17: l-6. Welinder H, Nielsen J, Bensryd I, Skerfving S IgG antibodies against polyisocyanates in car painters. Clin Allergy 1988; 18:85-93. 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. Chen SE, Bernstein IL. The guinea pig model of diisocyanate sensitization. I. Immunologic studies. J ALLERGY CLIN IM-
to occupational
sensitizers
MUNOL 1982;70:383-92. 28. Patterson R, Zeiss CR, Harris KE. Immunologic and respiratory responses to airway challenges of dogs with toluene diisocyanate. J ALLERGY CLIN IMMUNOL 1983;71:604-11. 29. Tse KS, Chan H, Chart-Yeung M. Specific IgE antibodies in workers with occupational asthma due to western red cedar. Clin Allergy 1982;12:249. 30. Fabbri LM, Boschetto P, Zocca E, et al. Bronchoalveolar neutrophilia during late asthmatic reactions induced by toluene diisocyanate. Am Rev Respir Dis 1987;136:36-42. 31. de Monchy JGR, Kauffman HF, Venge P, et al. Bronchoalveolar eosinophilia during allergen-induced late asthmatic reactions. Am Rev Respir Dis 1985;131:373-6. 32. Lam S, Leriche J, Phillipps D, Chart-Yeung M. Cellular and protein changes in bronchial lavage fluid after late asthmatic reaction in patients with red cedar asthma. J ALLERGY CLIN IMMLJNOL1987;80:44-50.
Clinical and immunologic evaluations reactive dye-exposed workers
of
Hae Sim Park, MD, PhD,*t *** Mi Kyung Lee, MD,* Byung Ok Kim, MD,* Kyung Jong Lee, MD,** Jae Hoon Roh, MD, PhD,** Yong Han Moon, MD, PhD,** and Chein-Soo Hong, MD, PhD* Seoul, Korea To evaluate type I hypersensitivity to reactive dyes, its prevalence, and its relationship to respiratory dysfunction, we studied clinical and immunologic features, including skin prick tests, RAST, and bronchoprovocation tests, of 309 employees working in a reactive-dye industry. Our survey revealed that 78 (25.2%) employees had work-related lower respiratory symptoms associated with or without nasal, skin, or eye symptoms. Among these employees, 38 (48.7%) had nonspect$c bronchial reactivity. Thirteen demonstrated immediate (6) dual (6), or late only (1) asthmatic responses after inhalation of four kinds of reactive-dye solutions. Twenty-five employees demonstrated immediate skin responses to black GR dye, and 21 reacted to orange 3R. Fifty-three employees (17%) had spectfic serum IgE antibody against black GR and orange 3R-human serum albumin conjugate. Specific IgE was detected more frequently in symptomatic employees (30%) and smokers (100%). No association was found between atopy and specific IgE binding. The RAST-inhibition tests of black GR revealed sigmficant inhibitions by black GR-human serum albumin conjugate and minimal inhibitions by unconjugated black GR. Orange 3R RAST-inhibition tests revealed signi$cant inhibitions by conjugated forms of black GR and orange 3R and some inhibitions by two unconjugated dyes, suggesting an immunologic cross-reactivity between these dyes. These findings suggested that reactive dyes could induce immunologic responses, most likely IgE-mediated. (J ALLERGY CLINIMMUNOL1991;87:639-49.)
From the *Department of Internal Medicine and **Department of Preventive Medicine and Public Health, Yonsei University College of Medicine, Seoul, Korea. Received for publication Sept. 20, 1989. Revised Sept. 4, 1990. Accepted for publication Oct. 4, 1990.
Reprint requests: Chein-Soo Hong, MD, Department of Internal Medicine, Yonsei University College of Medicine, CPO Box 8044, Seoul, Korea. ***Current address: Hae Sim Park, MD, PhD, Department of Chest Medicine, National Medical Center, Jung-ku Eulgiro 6-ka 1879, Seoul, Korea. l/1/26272
640
Park et al
Reactive dyes that bind covalently to textiles have been used increasingly as coloring agents in the textile industry, and cases of occupational asthma caused by these dyes have been reported.lm3Positive skin tests and the presence of specific serum IgE antibodies to reactive dyes suggest that respiratory symptoms provoked by reactive dyes could be IgE-mediated reactions. Previously, we reported nine cases of occupational asthma associated with the exposure to three different reactive dyes.’ Docker et a1.4 reported 2 1 cases of occupational asthma associated with dye exposure among >400 workers. To evaluate type I hypersensitivity to reactive dyes, its prevalence, and its relationship to respiratory dysfunction, we performed SPTs and RAST to detect specific IgE antibodies to reactive dyes in 309 exposed workers of a reactive-dye industry. We also performed methacholine bronchial challenge tests and bronchoprovocation tests with reactive dyes in symptomatic employees. RAST-inhibition tests were performed to investigate the binding specificity of IgE antibodies and the cross-allergenicities between reactive dyes. MATERIAL Subjects
AND METHODS
All employees of the Ehwa Dye Industry, Incheon, Korea, 275 male and 34 female employees between the ages of 19 and 63 years (mean age, 39.8 years), working in the dye industry and exposed to all the dyes, were included in our study. Of these employees, 271 were dye-process workers who crushed and mixed the materials as well as dried and carried them. Seven employees were office workers, and staff of all the laboratory was 31. Of all the reactive dyes, black GR and orange 3R were the chemicals handled most frequently.
Questionnaires Initially, doctors administered a questionnaire, which was a National Heart and Lung Institute modification of the British Medical Research Council questionnaire,’ with additional questions later at each visit. In evaluating and comparing these data, the following definitions were used: Lower respiratory symptoms referred to cough, sputum, chest tightness, or shortness of breath. Symptomatic employees were those workers who had suffered from lower respiratory symptoms during and after the dye exposure.
Sera Sera from 309 employees were collected and stored at - 20” C, as well as sera from 11 individuals who had never been exposed to reactive dyes and who demonstrated negative skin tests to 50 common inhalant allergens as control subjects.
lie.-.-’ ^” --->-’
“..., . *,
_’ ,A’!
.~ ‘,. &,
‘)’
1
Abbreviations used HSA: Human serum albumin PC,,,: Provocation concentration! reql~ir;‘tI id. reduce FEV, 20% below bascli!:t: A/H ratio: Allergen to histamine whcai IZ~:~! SPT: Skin prick test
Preparation
of dye solutions
for SPTs
Seven kinds of reactive dyes were prepared by ?he dye industry. Their trade names and color index number: were Rifacion orange HE 2G (O-20), Rifacion red HE 313, Rifacion navy blue HER, Rifafix yellow 3 RN, Rifafix red BBN, Rifazol brilliant orange 3R (O-16), and Rifazoi black GR (BK-5). Two red dyes (Rifacion red HE 3B and Rifafix red BBN), navy blue, and yellow 3 RN were not listed in the color index. The structures of the two reactive dyes (Rifazol black GR:black GR and Rifazol brilliant orange 3R:orange 3R), the most frequent sensitizers on SPTs in exposed workers, are illustrated in Fig. 1. Ten milligrams of each reactive dye was dissolved in 1 ml of modified Coca solution (NaCl, 9 gm; phenol crystal, 4 gm; and NaHCO.3. 2.9 gm, in 1000 ml of distilled water) containing 50% sterile glycerine for SPTs.
Preparation
of dye-HSA
conjugate
disks
To prepare the disk, 0.1 mg of each reactive dye was added to 2 mg/ml of HSA (Sigma Chemical Co., St. Louis, MO.) and dissolved in 100 mmol/L of Na2C03 (pH 1I .O) according to the previously described method.’ This solution was incubated while it was being stirred at 4” C for 18 hours and then dialyzed against 100 mmol/L of Na2CO3 at 4” C for 48 hours. These dye-HSA conjugate solutions were used in preparing RAST solid phases and also in RAST-inhibition tests. Disks of nitrocellulose filter paper (catalog No. HAHY 08250, 0.45 pm, Millipore Co.. Bedford, Mass.) uere inmersed into the dye-HSA conjugate solutions for 72 hours at 4” C and dried at room temperature for use in the subsequent RAST assay.
RAST assay All employees were tested with two reactive dyes (black GR and orange 3R-HSA conjugate disks), which were the most frequent sensitizers in SPTs according to the previously described method.’ The dried disks were blocked with 10% newborn calf serum for 1 hour and incubated with 50 ~1 of sera for 6 hours at room temperature. The disks were washed three times with 2.5 ml of 0.9% NaCl containing RAST washing-solution additives (Pharmacia, Uppsala, Sweden). Then, 50 ~1 of “‘I-labeled antihuman IgE (Pharmacia) was added to each disk and left for 18 hours at room temperature The disks were washed again, and the bound lZ51was measured with a gamma counter. All assays were performed in duplicate. The results were expressed as percent binding,
VOLUME NUMBER
87 3
Reactive
Black
GR
641
NH2
~N=N$&N=N~SO,.,H,,O,,,,
S03H
Orange
HhSOC2H402S
IgE in dye workers
(EIK-5)
OH H03SOC2H,OpS
dye-specific
3R
+N
S03H
( O-161
=N&JNHCOCH3
S03H FIG. 1. The chemical
structures
defined as a percentage of the added counts per minute bound to the reactive dye-HSA conjugate disks.
Measurement
of total IgE
The total IgE was measured with a Phadebas PRIST kit (Pharmacia) according to the manufacturer’s directions. One anti-IgE disk was put into the bottom of each tube and incubated with 100 ~1 of diluted serum at room temperature for 3 hours. The disks were washed three times with 2.5 ml of 0.9% NaCl containing PRIST washing additives. Then, 100 ~1 of anti-IgE ‘251-labeledPRIST tracer was incubated for 18 hours at room temperature and rinsed three times. The binding radioactivities were determined with a gamma counter, and the absolute amounts of the total IgE were determined on a standard curve.
RAST-inhibition
tests with
reactive
of the two reactive
dyes studied.
TABLE I. Number of SPTs and RAST positives to each reactive dye in 309 employees Reactive dyes
SPT (%I
RAST (“/.I
Orange HE 2G Red HE 3B Navy blue HER Yellow 3 RN Red BBN Brilliant orange 3R Black GR
1 (0.3) 1 (0.3) 1 (0.3) 3 (1.0) 3 (1.0) 21 (6.8) 25 (8.1)
ND ND ND ND ND 39 (12.6) 52 (16.8)
ND. Not done.
dyes
Competitive RAST inhibition was performed to investigate the specificity of the IgE binding to the dye-HSA conjugate as well as the relationship between the two reactive dyes according to the previously described method.’ The sera of seven patients who had high IgE bindings (>20% bound radioactivity) on dye-HSA conjugate disks were pooled. Of this pooled sera, 50 ~1 was incubated with four concentrations (0.0025 to 0.1 mg/ml) of reactive dyeHSA conjugates, unconjugated dyes, or HSA alone for 1 hour at room temperature. The sera pool was then incubated with black GR-HSA conjugate disks for 6 hours at room temperature. After washing three times with 2.5 ml of 0.9% NaCl containing RAST washing additives, 50 p,l of lz51labeled antihuman IgE (Pharmacia) was incubated with the disks for 18 hours at room temperature. After repeated wash-
ings, the bound radioactivities were determined with a gamma counter. After studying the control samples in which an equal volume of phosphate-buffered saline, pH 7.5, was preincubated instead of the inhibitors, the inhibition of the specific IgE binding was expressed as follows: cpm bound to dye-HSA disk with inhibitor 100 x 100 (%) cpm bound to dye-HSA disk with PBS
Allergy
skin test
SPTs with seven common inhalant allergens (oak, ryegrass, ragweed, wormwood, Aspergillus spp, Dermatopha-
642
Park et a!.
TABLE
II. Methacholine
Work-related
bronchial
challenge
test and RAST positives No. of employees (%.)
symptoms
Lower respiratory symptoms Respiratory symptoms alone Plus nasal symptoms Plus nasal plus skin symptoms Plus nasal plus eye symptoms Plus nasal plus eye plus skin symptoms Plus eye symptoms Plus skin symptoms Nasal symptoms Nasal symptoms alone Plus skin symptoms Skin symptoms alone Eye symptoms alone
78 (25.2) 38 (12.3) 28 (9.1) 2 (0.7) 3 (1.3) I (0.3) 2 (0.7) 3 (I .O) 27 (8.7, 26 (8.4) I (0.3) I (0.3) I (0.3)
in symptomatic
employees
Methacholine bronchial challenge test 1%)
3X/78 (48.7) 22138 (57.9) I?.:28 (42.8) 1/2 (50.0) 114 (25.0, O! 1 (0) 012 (0) 2i.3 (66.7) ND ND ND ND ND
ND, Not done. Lower respiratory symptoms: cough, sputum, and shortness of breath. Nasal symptoms: rhinorrhea, sneezing, and congestion. Skin symptoms: urticaria and pnnitus.
goides furinae, cat fur, Bencard Allergy Unit, Brentford, Middlesex, England), seven reactive dye solutions (10 mg/ml), and histamine (1 mg/ml, Bencard Allergy Unit) were performed on the volar side of both forearms simultaneously. The reactions were read 15 minutes later. The wheal size of each antigen (A) and histamine (H) was measured by the maximum diameter (A 1 and H 1) and vertical length at the midportion of the maximal length (A2 and H2). Skin reactivity was expressed as the ratio of the wheal size of the antigen to that of the histamine Al + A2/H 1 + H2 A/H ratio). According to the criteria of the Scandinavian Society of Allergology,b if the A/H ratio is from 0.1 to 1 and erythema with a diameter is 21 mm, it is read as 2 + A positive responder was defined as a responder who demonstrated more than a 2 + response to any reactive dye solution on SPT.
Methacholine
bronchial
challenge
test
Symptomatic employees identified by our questionnaire were referred to the Allergy Clinic of Severance Hospital, Seoul, Korea. Of this group, employees demonstrating nonspecific bronchial reactivity was determined by the previously described standard method.’ An aerosol of 0.9% NaCl, followed by serial doubling concentrations of methacholine (0.075 to 25 mg/ml), was inhaled. The FEV, was measured 5 minutes after each inhalation until the FEV, had fallen by 20% from the postsaline basal value. It was determined that nonspecific bronchial reactivity was present if a patient demonstrated more than a 20% decrease in FEV, after inhalation of any concentration (0.075 to 2.5 mg/ml) of methacholine. PC, was obtained from the dose-response curve.
Bronchoprovocetion
test& w&h reeetive
Qves
Bronchoprovocation tests were performed in an open manner without any placebo control according to the previously described method’ in 20 employees with asthma whose methacholine PC,, was 2 + of an A/H ratio to black GR, and 21 (6.8%) reacted to orange 3R. However, there were few responders to
VOLUME NUMBER
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Reactive
dye-specific
IgE in dye workers
643
TABLE III. The results of SPTs, RAST, and bronchoprovocation tests in patients with asthma who demonstrated a positive response to bronchoprovocation tests with reactive dyes SPT (A/H ratio)
Patient
Orange HE 26
S. K.
0
Y.S. K. J. K. Y. L. Y. P. K. M. H. C.J. C.O. P. B. C. E. P. J. c. Y.
0 0 0 0 1.4 0 0 0 0 0 0 0
RAST (bound
Yellow 3 RN
Red BBN
Orange 3R
Black GR
2.5 0.75 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0.44 0 0 0
3.5 0 0 0.83 0.8 0.8 0.6 0 1.33 1.33 0 0 0
0 0 1.25 0 0.1 0 0 0.9 1.33 0.56 0 0 0
Orange HE 2G
ND ND ND ND ND 0.8 ND ND
ND
%)
Bronchoprovocation
Yellow 3 RN
Red BBN
Orange 3R
Black GR
1.6 2.3 ND ND ND ND ND ND ND
ND ND ND ND ND ND ND ND ND
1.0 0.5 0.4 13.0 1.5 0.6 9.0 14.7 4.04 7.2 0.63 0.74 22.74
1.0 0.7
ND
ND
3.1
ND ND ND
ND ND ND
ND ND ND
0.8
15.3 1.1 0.9 6.4 28.6 6.75 14.4 0.59 2.47 16.65
Antigen
Yellow 3 RN Yellow 3 RN Black GR Orange 3R Black GR Orange HE 2G Orange 3R Black GR Orange 3R Orange 3R Orange 3R Black GR Black GR
test
Response
Dual Early Early Dual Dual Dual Dual Dual Early Early Late only Dual Early
A/H ratio: wheal size of dyes to that of histamine.
orange HE 2G (I), red HE 3B (l), navy blue HER (l), yellow 3 RN (3), and red BBN (3) (Table I). The questionnaire data are summarized in Table II. Seventy-eight (25.2%) employees complained of lower respiratory symptoms with or without nasal, skin, or eye symptoms during and after dye exposure. Twenty-six (8.4%) employees had nasal symptoms only. When methacholine bronchial challenge tests were performed on 78 employees with lower respiratory symptoms, 38 (48.7%) had nonspecific bronchial reactivity. Bronchoprovocation tests with four different reactive dyes were performed on 20 symptomatic employees whose methacholine PC,, was 2%, 53 (17%) of 309 employees were positive, 52 (17%) to black GRHSA conjugate, and 39 employees (13%) to orange 3R-HSA conjugate. Among the 13 cases that demonstrated positive results on the reactive-dye bronchoprovocation test, eight demonstrated high specific IgE antibodies to the corresponding reactive dye (Table III). The relationship between the specific IgE antibodies to black GR and orange 3R-HSA conjugate and cigarette smoking is presented in Table IV. A statistical significance was found between the two (p < 0.05). When atopy was defined as a positive reactor (A/H ratio 2 2 +) to one or more allergens on SPTs with seven common inhalant allergens, no association was found between atopy and specific IgE antibodies to black GR or orange 3R-HSA conjugate (Table V). To assess the influence of total IgE level on specific IgE binding, RAST to black GR and orange 3R were performed with sera from individuals who had a high total IgE level (700 to 12000 IU/ ml) and who had never been exposed to reactive dyes. The bound radioactivities on two dye-HSA disks were all < 1% as illustrated in Fig. 4. Total IgE level A high total IgE level (> 160 IU/ ml), which was derived from the mean and 2 SD of the total IgE level in nonatopic patients in Korea’ were found in 142 (46%) of all those employees who were tested. The correlation between the total IgE level and exposure duration was poor (r = 0.07). The correlation coefficient between the total IgE level and specific IgE
644
Park et al.
. 40-
. C 8 ,o g
30.
‘Z s
. . .
(n*231) FIG. 2. The specific subjects.
IgE binding
to black GR-HSA conjugate
antibodies was 0.39 in black GR (Fig. 5) and 0.36 in orange 3R (data was not presented). Specifm IgE to other reactive dye-WA conjugates The RAST results to other reactive dyes in nine positive reactors on SPTs are presented in Table VI. Seven workers had high specific IgE antibodies to these dye-HS A conjugates.
(n=ll) in exposed
RAST inh&itbn
employees
and control
tests
The dose-response pattern of the inhibitory effect of various preparations on the black GR RASTinhibition test is illustrated in Fig. 6. The RAST percent bindings for the uninhibited set-a were 19.7% (black GR) and 13.3% (orange 3R). Black GR-HSA was the most effective inhibitor. Unconjugated form of black GR and orange 3R-HSA conjugate resulted in some inhibitions. An unconjugated form of -orange
VOLUME NUMBER
87 3
Reactive
dye-specific
IgE in dye workers
645
.
(nm78) FIG. 3. The specific subjects.
IgE binding
(nm 231)
to orange 3R-HSA conjugate
3R and HSA alone led to no inhibition. The orange 3R RAST-inhibition test revealed significant inhibitions by both black GR-HSA and orange 3R-HSA conjugate. Two unconjugated dyes resulted in some inhibition. HSA alone elicited no inhibitions as illustrated in Fig. 7. DISCUSSION
Workers in dye industries are exposed to various respiratoryhazards.The major allergenic sourceshave
in exposed
employees
and control
been known to be reactive dyes.4 In this study, we demonstratedthe clinical and immunologic responses of workers exposed to reactive dyes. Seventy-eight (25.2%) of 309 employeestestedhad pronouncedrespiratory symptoms. Among them, 23 (29.5%) employees had specific serum IgE antibodies to black GR-HSA conjugate and 36 (48.7%) had nonspecific bronchial reactivity. Inhalation challenge tests with reactive dyes induced immediate asthmaticresponses in six employees, a late responsein one employee,
846
Park et al.
s
2.0-
z .-> t
4
38
l.O- -
a
z ci m
.-
Block- GR
+----*
Orange 3R
ffQqQ?:+//4=: ,’
v 0
1 0
1
,
2000
f 4000
I
I 6000
1
Total IgE Level FIG. 4. The influence
I 8000
I
I IO000
of total IgE on black GR and orange
3~ RAST,
.
I
4
6
8 101214
Specific FIG. 5. The correlation
between
I
I
I
I,,
,
,
,
. r = 0.39
.
2
f
(IU/mi)
1
0
I
,
,,
16182Q222428283032343636404244
IgE AnNbodiesto Black GR-HSA t&jtotal IgE and specific IgE antibodies
and dual asthmatic responsesin six employees. Severa1 mechanisms may be involved in reactive-dye asthma. In patients with specific IgE antibodies, the IgE-mediated reaction is likely to be responsible for their asthmatic symptoms. In this study, eight of 13
(IkundK)
to black GR-HSA conjugate.
patients with asthma who demonstratedpositive responseson the bronchoprovocationtest with r~ctive dyes had high levels of specific IgE antibodies to reactive dye-HSA conjugate. The failure to detect specific IgE antibodiesto reactive dyes in the seraof five
VOLUME NUMBER
87 3
100 1
Reactive
dye-specific
IgE in dye workers
647
Block GR- RAST Orange 3R- RAST
Black
Block
GR-H$
,
.
Conjugate
GR-l-ISA /
IOFree Oronqe O.CO25
0.01 Inhibitors
0.025
3R
HSA Alone
?
0. I Added
o0.002s
0.01
ISA
0.025
0.1
T 0.5
0.2
Alone * T I.0 1.0 2.0
Inhibitor8 Added (mg/ml)
(mq/ml)
FIG. 6. Percent inhibition of black GR-RAST with addition of black GR and orange 3R-HSA conjugate, free black GR, orange 3R, and HSA alone.
FIG. 7. Percent inhibition of orange 3R RAST with the addition of black GR and orange JR-HSA conjugate, free black GR, orange 3R. and HSA alone.
TABLE IV. The association and specific IgE antibodies orange BR-HSA conjugate
TABLE V. The relationship between atopy and specific IgE antibody to black GR and orange 3R-HSA conjugate
between smoking to black GR and
Specific
Nonsmoker Former smoker Current smoker
IgE antibodies
(%)
Black GRl-ISA*
Orange 3RHSAt
2194 (2) 6/46 (13) 46/ 167 (28)
3194 (3) 3146 (7) 37/ 167 (22)
*Significant differences were found @ < 0.05). Significant differences were found @ < 0.05).
workers with asthma might indicate the involvement of another immunologic or nonimmunologic mechanism in the induction of their asthmatic symptoms. Since the reactive-dye bronchoprovocation test was performed in an open manner with no placebo control, the possibility of false-positive responses caused by psychogenic factors, nonspecific irritating effects, or to natural variability of asthma cannot be completely exluded. However, our previous study” revealed that 10 workers with asthma demonstrated negative responses on black GR bronchoprovocation test among
Specific
IgE antibodies
(%)
Black GRHSA
Orange 3RHSA
Presence
29/ 182 (16)*
221182 (12)‘t
Absence
23/121 (19)*
19/ 121 (16)t
Atow
*p X0.05, compared between two groups. tp > 0.05, timpared between two groups.
TABLE VI. The RAST positive to five other reactive dyes in positive reactors on SPTs with reactive dyes Reactive
dyes
Orange HE 2G Red HE 3B Navy blue HER Yellow 3 RN Red BBN
Patients tested
1 1 1 3 3
No. (%I
0 (0) 1 (100.0) 1 (100.0) 2 (66.7) 3 (100.0)
648 Park et al 30 workers with asthma challenged by black GR. Three of 30 workers with asthma demonstrated dual asthmatic responses at low concentrations of reactive dye, suggesting that their asthmatic symptoms were caused by immunologic reactions. Among 13 positive responders on the bronchoprovocation test, four employees with asthma demonstrated positive SPTs but negative RAST. In these cases, another immunologic mechanism involving IgG has been suggested to have a role in the pathogenesis of their asthma.” Although the significance of the IgG, short-term sensitizing, antibody-mediated reaction in occupational asthma is unknown, it might be possible that this alternate mechanism could occur in a situation in which clinical sensitivity is suspected but specific IgE antibodies cannot be detected. Most patients with symptomatic occupational asthma have been known to have demonstrable nonspecific bronchial reactivity.” However, it is uncertain at the present time whether the bronchial reactivity is the result of occupational exposure or a predisposing factor. Lam et al.‘* suggested that nonspecific bronchial hyperreactivity in red cedar asthma was a result of exposure rather than a predisposing factor. In addition, increased airway responsiveness occurs after exposure to occupational and environmental agents.“16 The presence of preexisting bronchial hyperreactivity before employment can be an additional risk factor for occupational asthma. Brooks et al.” suggested that airway inflammation caused by inhaled toxic agents was responsible for the change in histamine responsiveness. There are some studies”. I9 documenting the inflammatory nature of various reactions to irritants. It might be possible to ascertain these suggestions if prospective studies of workers are performed, including preemployment methacholine bronchial challenge tests and regular follow-ups. Additional investigations are needed to clarify the combination effect of allergenic substances and irritant vapors, such as sulfur dioxide and hydrochloric acid, used in the dye process. We used RAST to detect specific IgE to dye-HSA conjugates. We were confronted with an enormous number of reactive dyes, many of whose chemical structures had not been published. Black GR-HSA conjugate was demonstrated to be by far the most sensitive antigen. Thirty-one of 52 sensitized employees to black GR and 14 of 39 sensitized employees to orange 3R had specific IgE binding on RAST but gave negative responses on skin tests. These discrepancies may have occurred because the SPTs were done with unconjugated dyes. Positive skin tests and the presence of specific IgE antibodies indicated sensiti-
J ALLERGY
CLl?l. :MMUNOL. MARCH 1991
zation. Twenty-six employees found to have high specific IgE binding did not have any symptoms. Additional investigations are needed to evaluate the ciirncal significance of these asymptomatic cemihzed workers. The striking finding of this study was the a%sociation between smoking and specific IgE production: which is a comparable finding to some other investigators.‘“, ‘I Although the role of cigarette smoking in the development of occupational sensitization is unknown, a possible explanation is that the lungs of smokers might be more permeable to inhaled antigens,” presumably the result of the increased permeability of the bronchial epithelium caused by cigarette smoking.” On the contrary, no association was found between atopy and specific IgE antibodies in this study. Other investigators have also noted that in industries in which low molecular weight compounds were responsible, such as western red cedai’ and isocyanate manufacturers,” atopy was not a predisposing factor. In most cases of occupational asthma induced by low molecular weight compounds. the compounds act as haptens and combine with a protein carrier to act as allergens, 21In contrast, isocyanates, such as toluene diisocyanate and hexamethylene diisocyanate, by virtue of their high degree of reactivity, appear to alter the native structure of the carrier protein to the extent that both haptenic and new antigenie determinants could be determined.‘6. ” In the black GR RASTinhibition test, significant inhibition was found with black GR-HSA conjugate and negligible inhibitions by unconjugated black GR, orange 3R-HSA conjugate, and an unconjugated form of orange 3R. Orange 3R RAST-inhibition tests demonstrated that significant inhibitions were found with the addition of black GR-HSA conjugate and unconjugated black GR, as well as with orange 3R-HSA conjugate and the unconjugated form of orange 3R, suggesting crosbreactivity between these two dyes. It is possible that the apparent cross-reactivity could be due fo shared epitopes of the new antigenic determinants because these two dyes are structurally very similar. In conclusion, we suggest that reactive dyes can induce an immunologic, mostly &E-mediated, response in dye-exposed employees, which is responsible for their asthmatic symptoms. The specificity of IgE binding on dye-HSA disks might involve new antigenic determinants or haptenic mechanisms. Additional investigation is needed to clarify other immunologic or nonimmunologic mechanisms involved and the prognostic significance of asymptomatic sensitized employees.
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