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Analysis of Antigens in Cotton Dust by Immunological Methods ANTONIO A . SEKUL and ROBERT L . ORY U.S.Department of Agriculture, Agricultural Research Service Southern Regional Research Center, New Orleans, L A 70179

Aqueous extracts o f cotton dust and cotton bract induced the formation o f specific precipitating antibodies i n rabbits. The a n t i s e r a c r o s s - r e a c t e d with both extracts as well as with extracts o f cotton stem, leaf, and burr, baled cotton and gin trash. Cross-reactivity was also demonstrated with extracts of flax, soft hemp, sisal, and jute. No antigen-antibody reaction was obtained with extracts o f cottonseed hulls, cottonseed p r o t e i n s , noncontaminated cotton lint, or house dust. No reaction was obtained between the a n t i s e r a to dust and several commercial preparations o f bacterial lipopolysaccharides believed t o be present i n cotton dust. With the exception o f extracts from burr, none o f the antigens reacted with preimmunization serum. Dust generated i n cotton t e x t i l e processing plants i s a hazard f o r some workers. Those that are susceptible and are exposed to cotton dust f o r a long period of time may be affected by a symptoms-complex known as byssinosis (1), a syndrome characterized by periodic coughing, wheezing, chest tightness, and decreased pulmonary function (2,3). The symptoms are more obvious on the f i r s t day back after a rest from exposure (Monday phenomenon). Prolonged exposure to the dust may cause i r r e v e r s i b l e respiratory impairment. The disease has also been described in workers in the flax and soft hemp industries (4,5,6), as well as in the sisal and jute industries (7-10). In its acute stage as well as in i t s chronic stage, byssinosis remains a major problem in the vegetable fiber processing industry. Byssinosis, however, i s not understood i n either i t s mechanism or specific etiology (11). Accumulated evidence shows that in cotton m i l l s , byssinosis is not caused by the cotton l i n t i t s e l f , but rather by foreign matter in the dust arising from the processing of raw fibers (12). This chapter not subject to U.S. copyright. Published 1982 American Chemical Society.

Montalvo; Cotton Dust ACS Symposium Series; American Chemical Society: Washington, DC, 1982.

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C o t t o n dust i s a heterogeneous m i x t u r e o f v a r i e d c o m p o s i t i o n (13), but c o t t o n b r a c t has been i d e n t i f i e d as one o f the major c o n t a m i n a n t s (32-52%) o f the p l a n t t r a s h (14.). Many d i f f e r e n t agents and t h e o r i e s have been p o s t u l a t e d t o e x p l a i n the causes o f b y s s i n o s i s . F o r example, p r o t e o l y t i c enzymes d e r i v e d from c o t t o n s e e d p r o t e i n s , b a c i l l i , and f u n g i , are thought t o be a s s o c i a t e d w i t h the d i s e a s e (15). Numerous r e p o r t s i m p l i c a t e h i s t a m i n e o r h i s t a m i n e l i b e r a t o r s ~ ~ ( 2 , 16-19), m e d i a t o r s o f smooth muscle s t i m u l a n t s (11,20), and p h a r m a c o l o g i c a l l y - a c t i v e s u b s t a n c e s such as l a c i n i l e n e C-7 methyl e t h e r , which causes l e u k o c y t e chemotaxis i n v i v o and i n v i t r o (21). The p o s s i b i l i t y that gram-negative microorganisms and/or t h e i r l i p o p o l y s a c c h a r i d e s (LPS) may have a r o l e i n p r o d u c i n g b y s s i n o s i s has been s t u d i e d w i t h g r e a t i n t e r e s t . S e v e r a l i n v e s t i g a t o r s have demons t r a t e d t h a t a i r b o r n e g r a m - n e g a t i v e b a c t e r i a i n c o t t o n m i l l s are c l o s e l y a s s o c i a t e d w i t h t h e p r e v a l e n c e o f b y s s i n o t i c symptoms among w o r k e r s , but the e x a c t r o l e o f t h e s e m i c r o o r g a n i s m s i n the e t i o l o g y o f the d i s e a s e i s not c l e a r (22-25). O t h e r workers (9., 16) s t a t e d t h a t the a c t i o n o f c o t t o n dust e x t r a c t s was not t y p i c a l o f e n d o t o x i n s (LPS) and t h a t b a c t e r i a l e n d o t o x i n s were not p r e s e n t i n the dust i n q u a n t i t i e s s u f f i c i e n t t o be e f f e c t i v e . Some i n v e s t i g a t o r s have suggested t h a t a n t i g e n i c m a t e r i a l p r e s e n t i n c o t t o n dust may p l a y an important r o l e i n the d i s e a s e (26,27). A phenolic polymer 5,7,3'4'-tetrahydroxyflavan-3,4 d i o l i s o l a t e d from c o t t o n b r a c t was thought t o be the r e s p o n s i b l e agent (28). T h i s polymer was l a t e r found t o r e a c t w i t h a l l human s e r a , w i t h s e v e r a l myeloma IgG p r e p a r a t i o n s , and w i t h the i s o l a t e d F c p i e c e o f IgG on gel d i f f u s i o n (29,30). R e c e n t l y , o t h e r r e p o r t s have appeared (31), as well as our own s t u d i e s (32), showing t h a t c o t t o n dust a c t i v a t e s the a l t e r nate pathway o f complement. P r e v i o u s s t u d i e s i n t h i s l a b o r a t o r y (33,34), showed t h a t a n t i b o d i e s t o water e x t r a c t s o f c o t t o n d u s t , c o t t o n b r a c t , and a h i g h l y p u r i f i e d f r a c t i o n o f d u s t e l i c i t e d p o s i t i v e immunological responses i n r a b b i t s . T h i s r e p o r t d e s c r i b e s our r e s u l t s on a n a l y s e s o f a n t i g e n s i n c o t t o n dust and b r a c t by immunological t e c h n i q u e s , and d i s c u s s e s t h e p o s s i b i l i t y o f a p p l y i n g t h e s e r e s u l t s t o t h e development o f a b i o a s s a y f o r a c t i v e agents which may be r e l a t e d t o b y s s i n o s i s . M a t e r i a l and Methods P r e p a r a t i o n o f Aqueous E x t r a c t o f C o t t o n D u s t . C o t t o n cardroom dust was c o l l e c t e d from V - c e l l f i l t e r s i n a commercial t e x t i l e m i l l . A t y p i c a l e x t r a c t i o n was c a r r i e d out by m a n u a l l y k n e a d i n g 50 g o f dust w i t h 500 ml o f d e i o n i z e d w a t e r f o r 5 min a t 25°c and removing the l i q u o r by c e n t r i f u g a t i o n . T h e p r o c e s s was r e p e a t e d t w i c e w i t h 250 ml o f d e i o n i z e d water each t i m e . T h e combined s u p e r n a t a n t was f i l t e r e d t h r o u g h f i l t e r paper by g r a v i t y and the f i l t r a t e ( o f f i n a l pH 8.3 w i t h o u t a d d i t i o n o f b u f f e r ) was f r e e z e - d r i e d t o y i e l d f r a c t i o n 1 ( f - 1 ) . The major p o r t i o n o f f - 1


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(lOg) was e x t r a c t e d t h r e e t i m e s w i t h 50 ml p o r t i o n s o f 85% methanol at 25°c f o r 30 min each t i m e w i t h s t i r r i n g then f i l t e r e d . The combined s o l u t i o n was e v a p o r a t e d t o dryness under r e d u c e d p r e s s u r e and l a b e l e d f r a c t i o n 2 ( f - 2 ) . T h e m e t h a n o l - i n s o l u b l e m a t e r i a l was l a b e l e d f r a c t i o n 3 ( f - 3 ) . F i v e g o f f-3 was d i s s o l v e d i n 50 ml o f water and t r e a t e d g r a d u a l l y with an e x c e s s o f l e a d a c e t a t e (20 ml; 0.2M), w i t h v i g o r o u s s t i r r i n g . The l e a d a c e t a t e s o l u b l e m a t e r i a l was t r e a t e d by s l o w l y adding 100 ml o f a 0.4M s o l u t i o n o f sodium s u l f a t e ( e x c e s s ) w i t h s t i r r i n g and f i l t e r e d . The f i l t r a t e was d i a l y z e d f o r 72 h r at 4°c a g a i n s t s e v e r a l changes o f d e i o n i z e d w a t e r . The s o l u t i o n was then f r e e z e - d r i e d t o y i e l d a p u r i f i e d f r a c t i o n 4 ( f - 4 ) . The l e a d a c e t a t e i n s o l u b l e f r a c t i o n was t r e a t e d w i t h sodium s u l f i d e and the r e s u l t i n g s o l u t i o n was f r e e z e - d r i e d a f t e r d i a l y s i s t o o b t a i n f r a c t i o n 5 ( f - 5 ) . F r a c t i o n s f - 1 , f - 3 , f - 4 , and f-5 were used t o immunize r a b b i t s . P r e p a r a t i o n o f Aqueous E x t r a c t o f C o t t o n B r a c t . F i e l d - d r i e d c o t t o n b r a c t , 10 g, p i c k e d d i r e c t l y from p l a n t s i n a f i e l d n e a r Lake P r o v i d e n c e , L o u i s i a n a , was ground w i t h m o r t a r and p e s t l e , e x t r a c t e d t w i c e w i t h 100 ml o f d e i o n i z e d water at pH 7 f o r 30 min each t i m e at 25°c w i t h s t i r r i n g . The e x t r a c t s were combined and the s o l u t i o n was f i l t e r e d ; t h e f i l t r a t e was f r e e z e - d r i e d . T h i s b r a c t e x t r a c t (bAg) was a l s o used t o immunize r a b b i t s . A d d i t i o n a l bAg was f u r t h e r p u r i f i e d by t r e a t m e n t w i t h 85% methanol i n a s i m i l a r manner as w i t h c o t t o n d u s t . Preparation of Other Antigenic M a t e r i a l s . Cotton plant t i s s u e s (stem, l e a f , b u r r ) , c o t t o n g i n t r a s h , b a l e d c o t t o n , c l e a n c o t t o n l i n t , b o t h hand p i c k e d i n the f i e l d and from p l a n t s grown i n the greenhouse, c o t t o n s e e d p r o t e i n s , c o t t o n s e e d h u l l s , house d u s t , and f l a x , s o f t hemp, s i s a l , and j u t e f i b e r s , were e x t r a c t e d w i t h d e i o n i z e d w a t e r . The p u r i f i c a t i o n p r o c e s s was, however, stopped t o c o r r e s p o n d t o f-3 (see F i g u r e 1 ) . B a c t e r i a l LPS used i n t h i s study were p u r c h a s e d f r o m Sigma Chemical Co., S t . L o u i s , M0., and were as f o l l o w s : E s c h e r i c h i a c o l i s e r o t y p e s 0.26:B6; 0127:B8 ( p r e p a r e d both by the p h e n o l i c and the TCA p r o c e d u r e s ) ; 0127:BB (by the butanol p r o c e d u r e ) ; S e r r a t i a m a r c e s c e n s , and S h i g e l l a f l e x n e r i . The LPS were s o l u b i l i z e d i n water and used as a n t i g e n i c m a t e r i a l s . P r e p a r a t i o n o f R a b b i t Serum and A n t i s e r u m . F o u r w h i t e New Zealand r a b b i t s (2-3 kg) were f i r s t b l e d t o o b t a i n normal serum (NS), then i n j e c t e d s u b c u t a n e o u s l y with a t o t a l o f 2.5 mg o f e i t h e r f - 1 , f - 3 , f - 4 , or f - 5 i n complete Freund's a d j u v a n t . A f t e r 10 d a y s , b o o s t e r i n j e c t i o n s o f the same amount o f a n t i g e n i n incomplete adjuvant were g i v e n e v e r y 2 weeks f o r a t o t a l o f seven i n j e c t i o n s . A n t i s e r a t o dust f r a c t i o n s (AD) and to b r a c t (AB) were pooled and c o n c e n t r a t e d a p p r o x i m a t e l y t e n f o l d by 40% s a t u r a t i o n w i t h ammonium s u l f a t e at 4°c. The p r e c i p i t a t e d


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Figure 1. Fractionation of cotton dust. Room temp, 25°C; dialysis time, 5 days.


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a n t i b o d i e s were resuspended i n a minimal amount o f 0.01M p h o s p h a t e - b u f f e r e d s a l i n e (PBS) a t pH 7.2 and d i a l y z e d e x h a u s t i v e l y a g a i n s t s e v e r a l changes o f PBS. NS was c o n c e n t r a t e d i n a s i m i l a r manner. AD, AB, and NS were used i n d o u b l e d i f f u s i o n experiments t o detect the a n t i b o d i e s . D e t e c t i o n o f A n t i b o d i e s . The method used f o r d e t e c t i n g a n t i b o d i e s was t h e d o u b l e d i f f u s i o n t e c h n i q u e i n a g a r o s e . Agarose p l a t e s ( 8 . 5 x 9.5 cm) were p r e p a r e d by p o u r i n g 10 ml o f a 1% molten s u s p e n s i o n o f a g a r o s e i n a b a r b i t a l b u f f e r o f 0.02 i o n i c s t r e n g t h and pH 8.6 ( 3 5 ) . A f t e r t h e g e l had s e t , c i r c u l a r w e l l s of 50 u l were punched out w i t h a c o r k b o r e r and f i l l e d once w i t h t h e a p p r o p r i a t e serum, a n t i s e r u m , o r a n t i g e n i c m a t e r i a l . T h e a n t i g e n s were a p p l i e d as 100 mg/ml s o l u t i o n s ( e x c e p t f o r b u r r , which was 25 mg/ml because o f i t s gummy c h a r a c t e r i s t i c and d i f f i c u l t y i n h a n d l i n g ) i n d e i o n i z e d water. D i f f u s i o n was a l l o w e d to proceed f o r 20-30 h r a t 25°c. P l a t e s were washed w i t h e i t h e r 0.2M sodium c h l o r i d e o r 0.15M sodium c i t r a t e f o r 24 h r p r i o r t o s t a i n i n g w i t h 0.5% s o l u t i o n o f amido b l a c k 10B. Chromatography and E l e c t r o p h o r e s i s o f A n t i g e n s . C o t t o n dust (20-30 mg) was d i s s o l v e d i n water and passed t h r o u g h a s m a l l column (30 x 1.5 cm i d ) packed w i t h 3 g o f Sephadex G-75 t h a t had been p r e s w e l l e d i n water. Chromatograms were o b t a i n e d w i t h an LKB U v i c o r d u l t r a v i o l e t m o n i t o r and a chopper b a r r e c o r d e r . Volumes o f 5 ml each were c o l l e c t e d i n t e s t tubes w i t h an a u t o m a t i c f r a c t i o n c o l l e c t o r . Two components were d e t e c t e d a t 250 nm. Samples o f 10 mg were a n a l y z e d by d i s c g e l e l e c t r o p h o r e s i s a c c o r d i n g t o p r o c e d u r e s d e s c r i b e d by D a v i s ( 3 6 ) , and Z a c h a r i u s (37). — Q u a n t i t a t i o n o f Human IgG. R a d i a l i m m u n o d i f f u s i o n t e c h n i q u e ( T r i - P a r t i g e n ; C a l b i o c h e m - B e h r i n g Corp.) was used t o measure n o r mal serum and serum from b y s s i n o t i c p e r s o n s . A f t e r f i l l i n g t h e w e l l s , d i f f u s i o n was a l l o w e d t o proceed f o r 50 h r a t 4°C. P r e c i p i t i n r i n g s were measured and t h e c o n c e n t r a t i o n o f IgG i n mg/dl was o b t a i n e d from t h e T a b l e o f R e f e r e n c e s s u p p l i e d w i t h t h e plates. R e s u l t s and D i s c u s s i o n F i g u r e 1 shows t h e f l o w s h e e t diagram f o r t h e f r a c t i o n a t i o n of c o t t o n d u s t . A c t i v e a n t i s e r a were o b t a i n e d w i t h f r a c t i o n s f - 1 , f - 3 , and f - 4 . F r a c t i o n 2 was not a c t i v e a g a i n s t a n t i b o d i e s to d u s t ( f - 1 ) , and f - 5 was i m m u n o g e n i c a l l y i n a c t i v e . In F i g u r e 2 d o u b l e d i f f u s i o n o f c o t t o n dust and b r a c t a n t i g e n s a r e shown a g a i n s t AD and NS. The c o n t i n u o u s l i n e s between bAg and f - 4 , and between f - 3 , and f - 4 i n d i c a t e a c r o s s r e a c t i o n o f f u l l i d e n t i t y i s p r e s e n t i n a l l t h r e e e x t r a c t s . S i m i l a r r e s u l t s were o b t a i n e d w i t h AB. F i g u r e 2 i n d i c a t e s t h a t a n t i g e n s i n c o t t o n dust and/or


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b r a c t do not r e a c t w i t h r a b b i t NS. T h i s d i f f e r s from the r e p o r t (29) t h a t c o t t o n dust r e a c t s w i t h both b y s s i n o t i c serum and normal serum. Both b r a c t a n t i g e n and a n t i g e n from c o t t o n dust r e a c t w i t h c o t t o n dust a n t i b o d i e s but t h e y do not r e a c t w i t h normal p r e - i m m u n i z a t i o n serum. T h i s i n d i c a t e s t h a t normal serum does not c o n t a i n a n t i b o d i e s t o e i t h e r b r a c t o r c o t t o n d u s t . These r e a c t i o n s d i f f e r from t h e pseudoimmune r e a c t i o n s r e p o r t e d by Edwards and J o n e s ( 3 0 ) , because the c o t t o n dust a n t i g e n ( s ) does not r e a c t n o n s p e c i f i c a l l y w i t h both normal and immunized r a b b i t s e r a . P r e c i p i t i n a r c s i n a r e a c t i o n o f i d e n t i t y between a n t i g e n s i n b r a c t s and dust i n F i g u r e 2 c o n f i r m t h a t a n t i g e n s p r e s e n t i n dust are d e r i v e d a t l e a s t p a r t l y from c o t t o n b r a c t . T y p i c a l e l e c t r o p h o r e g r a m s s t a i n e d f o r p r o t e i n and f o r c a r b o h y d r a t e are shown i n F i g u r e 3. The a n t i g e n i n f-4 a c t u a l l y c o n s i s t s o f two m a t e r i a l s ; a l a r g e , slow moving, d i f f u s e band near t h e o r i g i n , and a much s m a l l e r , sharp band t h a t m i g r a t e s about t w o - t h i r d s o f the way down the g e l . To d e t e r m i n e the type o f m a t e r i a l p r e s e n t i n t h e bands, a dual s t a i n i n g t e c h n i q u e was employed. Coomassie blue was used t o s t a i n f o r p r o t e i n and the S c h i f f ' s reagent t o s t a i n f o r p o l y s a c c h a r i d e . The l a r g e r slow moving band near the o r i g i n s t a i n e d h e a v i l y f o r c a r b o h y d r a t e but l i g h t l y o r not a t a l l f o r p r o t e i n . The s m a l l e r , sharp, f a s t moving band s t a i n e d f o r p r o t e i n but v e r y l i g h t l y f o r c a r b o h y d r a t e . These r e s u l t s i n d i c a t e t h a t t h e a c t i v e m a t e r i a l i n f - 4 i s g l y c o p r o t e i n i n n a t u r e . When t h e s e s e p a r a t e d f r a c t i o n s were t e s t e d f o r a n t i g e n i c i t y a g a i n s t a n t i b o d i e s t o c o t t o n d u s t , i t was f o u n d t h a t both were a c t i v e . To d e t e r m i n e i f p l a n t t i s s u e s o t h e r than b r a c t c o n t a i n e d t h e s e a c t i v e a n t i g e n s , h a n d - p i c k e d stem, l e a f , and b u r r t i s s u e s were o b t a i n e d from growing c o t t o n p l a n t s , d r i e d , then ground and e x t r a c t e d a s d e s c r i b e d p r e v i o u s l y . F i g u r e 4a compares immunodiff u s i o n o f b u r r , g i n t r a s h , l e a f , stem, c o t t o n d u s t , and c o t t o n bract. Results i n d i c a t e that the a n t i g e n i c m a t e r i a l s present i n c o t t o n dust and b r a c t i s a l s o p r e s e n t i n o t h e r p l a n t t i s s u e s (stem, l e a f , b u r r ) and i n g i n t r a s h c o l l e c t e d b e f o r e the c o t t o n e n t e r s t h e t e x t i l e m i l l cardroom. I m m u n o d i f f u s i o n t e s t s o f the same f r a c t i o n s a g a i n s t normal r a b b i t serum showed t h a t o n l y t h e e x t r a c t o f b u r r produced a very d i f f u s e p r e c i p i t a t e a g a i n s t normal serum ( F i g u r e 4 b ) . I t i s l i k e l y t h a t b u r r c o n t a i n s t h e common a c t i v e a n t i g e n found i n o t h e r t i s s u e s . However, t h e r e are o t h e r m a t e r i a l s , i n c l u d i n g a gummy e x t r a c t o f l a r g e m o l e c u l a r w e i g h t c a r b o h y d r a t e polymer, which are p r o b a b l y r e s p o n s i b l e f o r t h e o n l y i n s t a n c e o f n o n s p e c i f i c r e a c t i o n w i t h normal r a b b i t serum noted. The r e a c t i o n between b u r r e x t r a c t and dust a n t i s e r a does not s i g n i f y a g r e a t e r c o n c e n t r a t i o n o f a n t i g e n i n b u r r compared t o t h a t i n o t h e r e x t r a c t s . Except f o r the b u r r e x t r a c t , p r e c i p i t i n a r c s formed between a n t i g e n s i n o t h e r t i s s u e s and a n t i s e r a i n d i c a t e t h a t the a n t i g e n s i n the dust and p l a n t t i s s u e s are t h e same and t h a t t h e r e a c t i o n i s i r r m u n o l o g i c a l l y s p e c i f i c f o r a n t i g e n s common t o a l l . S i n c e b y s s i n o s i s has been r e p o r t e d


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Figure 2. Double diffusion of cotton dust fractions and cotton bract extract against rabbit antiserum to dust (AD) and normal rabbit serum (NS). Conditions described in Material and Methods.

Figure 3. Gel electrophoresis of cotton dust fraction 4 stained for protein (left) and carbohydrate (right). Conditions described in Material and Methods.


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Figure 4. Double diffusion of extracts from cotton plant tissues and extract from flax against rabbit antiserum to cotton dust. Key. a: AD, antisera to dust; gin, gin trash; If, leaf antigen; stm, stem antigen; cd, cotton dust antigen; bet, bract antigen; burr, burr antigen, b: NS, normal rabbit serum, c: fix, flax extract; f-4, cotton dust antigen. Conditions described in Material and Methods.


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t o o c c u r i n p e o p l e working w i t h o t h e r v e g e t a b l e t e x t i l e f i b e r s , we t e s t e d t h e r e a c t i o n o f a n t i b o d i e s t o dust w i t h e x t r a c t s o f f l a x , s o f t hemp, s i s a l , and j u t e . F i g u r e 4c shows a t y p i c a l r e a c t i o n o f e x t r a c t s from f l a x , f - 4 , and AD. In a l l i n s t a n c e s , r e a c t i o n s o f e x t r a c t s from v e g e t a b l e f i b e r s o t h e r than c o t t o n were p o s i t i v e w i t h AD but n e g a t i v e towards NS. There have been r e p o r t s o f r e a c t i o n s between house dust and c o t t o n dust a n t i b o d i e s (38) and o f a l l e r g e n s i n c o t t o n s e e d p r o t e i n s by S p i e s e t a 1 » T 3 9 ) . A l s o , b y s s i n o s i s i s uncommon i n c o t t o n s e e d c r u s h i n g m i l l s . T h e r e f o r e we l o o k e d f o r t h e p r e s e n c e o f a n t i g e n s i n water e x t r a c t s o f house d u s t , c o t t o n s e e d h u l l s , c o t t o n s e e d k e r n e l p r o t e i n s and c l e a n hand p i c k e d c o t t o n f i b e r s t h a t had not been b a l e d . The r e s u l t s i n F i g u r e 5 i n d i c a t e t h a t house d u s t does not c o n t a i n a n t i g e n s common t o t h o s e found i n cotton dust. R e s u l t s i n T a b l e I i n d i c a t e t h a t no immune p r e c i p i t i n r e a c t i o n o c c u r r e d between e x t r a c t s o f c o t t o n s e e d h u l l s , c o t t o n s e e d p r o t e i n , o r c l e a n c o t t o n f i b e r s ( p i c k e d from c o t t o n grown i n t h e greenhouse) and a n t i b o d i e s t o c o t t o n d u s t , b r a c t , o r normal serum. The r e a c t i o n i s p o s i t i v e t o b a l e d c o t t o n , however. The a n t i g e n s found i n c o t t o n d u s t o r b r a c t a r e n o t d e r i v e d from c o t t o n s e e d t i s s u e s , such as h u l l s o r c o t t o n s e e d p r o t e i n s , o r from c l e a n c o t t o n f i b e r s , but from c o t t o n p l a n t p a r t s o t h e r than t h e seed o r f i b e r . There have a l s o been r e p o r t s (24,26) t h a t p o s s i b l y b a c t e r i a l e n d o t o x i n s produced by m i c r o o r g a n i s m s growing i n c o t t o n , o r a s s o c i a t e d w i t h c o t t o n might be i m p l i c a t e d i n t h e p r o d u c t i o n o f a n t i b o d i e s . We c o n s i d e r e d LPS produced from c e r t a i n gram-negative organisms (some found on c o t t o n , some found i n s o i l ) f o r common a n t i g e n i c i t y t o a n t i b o d i e s i n c o t t o n d u s t . Data i n T a b l e 1 i n d i c a t e t h a t t h e s e p a r t i c u l a r LPS t e s t e d do not c r o s s - r e a c t with a n t i b o d i e s t o cotton dust. This suggests t h a t t h e s e p a r t i c u l a r e n d o t o x i n s a r e not a s s o c i a t e d w i t h t h e a n t i g e n i c m a t e r i a l s found i n c o t t o n dust o r b r a c t o r t h a t t h e i r c o n c e n t r a t i o n s were not h i g h enough t o be a f a c t o r i n t h e p r o d u c t i o n o f a c t i v e serum i n r a b b i t s . To determine whether normal humans who have not been a s s o c i a t e d w i t h t e x t i l e m i l l s produced immune r e a c t i o n w i t h a n t i b o d i e s t o c o t t o n dust o r b r a c t , 37 v o l u n t e e r s from t h e S o u t h e r n R e g i o n a l Research C e n t e r donated b l o o d . The b l o o d was c o l l e c t e d , t h e serum was s e p a r a t e d and c o n c e n t r a t e d a p p r o x i m a t e l y 7 - f o l d then t e s t e d f o r a n t i g e n - a n t i b o d y r e a c t i o n s . The m a j o r i t y produced no r e a c t i o n a t a l l but 5 d i d form what was r e f e r r e d t o as a n o n s p e c i f i c o r pseudoimmune r e a c t i o n . When b l o o d o b t a i n e d from known o r a l l e g e d b y s s i n o t i c s was compared w i t h t h e s e pseudoimmune-reacting b l o o d s e r a , t h e r e s u l t s showed ( F i g u r e 6a) that the material producing the antigen-antibody reaction i n n o r m a l s , does not c r o s s - r e a c t w i t h b l o o d from t h e b y s s i n o t i c s . However, t h e two known b y s s i n o t i c p a t i e n t s do show a r e a c t i o n o f i d e n t i t y (arrow; F i g u r e 6b) between t h e i r b l o o d s e r a and c o t t o n d u s t a n t i g e n ( c d ) . These two b l o o d samples were o b t a i n e d from


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Figure 5. Double diffusion of cotton dust and house dust extracts against rabbit antiserum to dust (AD). Key: f-3, cotton dust antigen; hd, house dust extract. Conditions described in Materials and Methods.


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Table I R e a c t i v i t y Between R a b b i t A n t i s e r a t o C o t t o n Dust and B r a c t , Normal R a b b i t Serum, and E x t r a c t s o f C o t t o n D u s t , C o t t o n T i s s u e s , House D u s t , and C e r t a i n B a c t e r i a l E n d o t o x i n s . Antigens

AD

AB

C o t t o n Dust Bract Stem Leaf Burr Gin Trash L i n t (Baled) Lint (Clean)

+ + + + + + +

+ + + + + + +

Cottonseed Proteins Hulls House Dust Endotoxins E. C o l i

026:B6 0127:B8 (Phenol E x t r a c t e d ) 0127:B8 (TCA E x t r a c t e d ) 0137:BB ( B u t a n o l E x t r a c t e d ) S e r r a t i a marcescens Shigella flexneri

(AD), a n t i s e r a t o d u s t ; ( A B ) , a n t i s e r a t o b r a c t ; (NS), normal serum; (+), p o s i t i v e r e a c t i o n ; (-), no r e a c t i o n .


NS

270

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Figure 6. Double diffusion of cotton dust antigen against human sera. Key: SRC 24, 37, and 16, normal human sera; RB and CJ, sera from byssinosis patients. Conditions described in Materials and Methods.


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b y s s i n o t i c p a t i e n t s e i g h t y e a r s a p a r t . One sample was h e l d i n a f r e e z e r a t -10°c and t h e o t h e r sample was f r e s h l y drawn. Nevert h e l e s s i t c o n f i r m s t h a t t h e s e two p e o p l e who had worked i n c o t t o n t e x t i l e m i l l s f o r many y e a r s , have a n t i b o d i e s t o a common c o t t o n d u s t a n t i g e n i n t h e i r b l o o d . They show a r e a c t i o n o f i d e n t i t y w i t h c o t t o n dust a n t i g e n . T h i s s u g g e s t s t h a t t h e a n t i g e n s which i n d u c e d f o r m a t i o n o f t h e s e a n t i b o d i e s i n t h e b l o o d o f t h e two p a t i e n t s a r e i d e n t i c a l . Kamat e t al_. (40) r e p o r t e d an e x a m i n a t i o n o f b l o o d serum immunoglobulins from c o t t o n m i l l workers h a v i n g b y s s i n o s i s o r c h r o n i c b r o n c h i t i s , and normal b l o o d serum. They showed t h a t normal p a t i e n t s had IgG l e v e l s a v e r a g i n g 1400 mg/dl, whereas b l o o d s e r a o b t a i n e d from b y s s i n o t i c s had a mean v a l u e o f 1850 mg/dl. In a v e r y l i m i t e d e x p l o r a t o r y way we examined b l o o d o f an SRRC normal p a t i e n t , a normal IgG s t a n d a r d o b t a i n e d from C a l b i o c h e m - B e h r i n g , and IgG l e v e l s from one o f t h e b y s s i n o t i c s . R e s u l t s i n F i g u r e 7 show t h a t t h e d i a m e t e r s o f t h e p r e c i p i t i n r i n g s around t h e two normal s e r a a r e e q u a l , but t h e d i a m e t e r o f t h e p r e c i p i t i n r i n g f o r t h e b y s s i n o t i c b l o o d i s much l a r g e r . U s i n g t h e t a b l e s u p p l i e d w i t h t h e s t a n d a r d IgG k i t , t h e normal had a c a l c u l a t e d IgG l e v e l o f 1292 mg/dl compared t o 1790 mg/dl for the b y s s i n o t i c blood. Summary R e a c t i o n o f a n t i s e r a t o c o t t o n dust and b r a c t a n t i g e n s produced p r e c i p i t i n a r c s which f u s e d i n a r e a c t i o n o f i d e n t i t y , i n d i c a t i n g common a n t i g e n i c d e t e r m i n a n t s i n t h e two m a t e r i a l s . C o t t o n d u s t , b r a c t , and a p u r i f i e d a n t i g e n s e p a r a t e d from t h e d u s t g i v e p o s i t i v e immune responses i n r a b b i t s . The immune response i s s p e c i f i c o n l y f o r r a b b i t s t h a t have been immunized w i t h dust o r b r a c t e x t r a c t s , not f o r normal r a b b i t serum. Sera o b t a i n e d from normal human v o l u n t e e r s do not c o n t a i n a n t i b o d i e s t o a n t i g e n s p r e s e n t i n c o t t o n dust o r b r a c t . S e r a o b t a i n e d from a l l e g e d b y s s i n o t i c p a t i e n t s do c o n t a i n a n t i b o d i e s s p e c i f i c f o r t h e s e a n t i g e n s . With a d m i t t e d l y l i m i t e d d a t a i t was i n d i c a t e d t h a t serum o b t a i n e d from an a l l e g e d b y s s i n o t i c had an i n c r e a s e d IgG l e v e l compared t o t h e IgG l e v e l i n normal b l o o d . A n t i g e n s found i n t h e d u s t and b r a c t a r e a l s o p r e s e n t i n l e a f , stem, and b u r r t i s s u e s o f t h e c o t t o n p l a n t , but not i n c o t t o n s e e d h u l l s , kernel p r o t e i n s , clean cotton f i b e r s , c e r t a i n b a c t e r i a l l i p o p o l y s a c c h a r i d e s o r house d u s t . The a n t i g e n i c m a t e r i a l was a l s o found i n o t h e r t e x t i l e f i b e r s such as s i s a l , s o f t hemp, j u t e , and flax. Disclaimer Mention o f company names o r p r o d u c t s does not c o n s t i t u t e endorsement by t h e U n i t e d S t a t e s Department o f A g r i c u l t u r e .


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Figure 7. Immunodiffusion of normal and byssinotic blood sera against human IgG antibodies. Tripatigen plate contains antibodies to human IgG in gel. Key: well 1, control IgG serum provided with plates; well 2, normal human serum; well 3, serum from byssinosis patient.


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A y e r , Howard E. Crit. Rev. E n v i r o n . Control 1971, 2, 207-41. Bouhuys, A. Trans. Ν . Y. Acad. Sci. 1966, 28, 480-90. Bouhuys, Α.; Schoenberg, J. B.; Beck, G. J.; Schilling, R. S. F . Lung 1977, 154, 167-86. Schilling, R. S. F . Lancet 1956, 2, 261-5-319-25. Bouhuys, Α.; Barbero, Α.; Lindell, S. E.; Roach, S. A. Arch. E n v i r o n . Health 1967, 14, 533-44. Z u s k i n , Eugenia; Valic, Fedor; Bouhuys, Arend. Lung 1976, 154, 17-24. Mustafa, K. Y.; Lakha, A . S.; Milla, M. H.; Dahoma, U . B r . J. Ind. Med. 1978, 3 5 , 123-8. Nicholls, P. J.; Evans, E.; Valic, F.; Zuskin, E. Br. J. Ind. Med. 1973, 30, 142-5. Nicholls, P . J. Br. J. Ind. Med. 1962, 26, 101-8. Popa, V.; Gavrilescu, N.; Preda,N.; Tesculescu, D . ; Plecias, M.; Cirstea, M. B r . J. Ind. Med. 1969, 26, 101-8. Battigelli, M. C.; Craven, P. L.; Fischer, J. J.; Morey, P . R.; Sasser, P. E., J . E n v i r o n . Sci. Health 1977, A12, 327-39. HEW p u b l i c a t i o n No. (NI0SH) 75-118, 1974, 159 pp. Wakelyn, P. J.; Greenblatt, G . Α.; Brown, D. F.; Tripp, V. W. Am. Ind. Hyg. Assoc. J. 1976, 37, 22-31. Morey, P . R.; S a s s e r , P . E.; Bethea, R. M.; Kopetzky, M. T . Am. Ind. Hyg. Assoc. J. 1976, 3 7 , 407-12. Braun, D. C.; S c h e e l , L . D.; Tuma, J.; Parker, L . J. Occup. Med. 1973, 15, 241-4. A n t w e i l e r , H. Br. J. Ind. Med. 1961, 18, 130-2. Edwards, J.; McCarthy, P.; McDermott, M.; Nicholls, P. J., Skidmore, J. W. J. Physiol. London 1970, 208, 63P-64P. Evans, E.; Nicholls, P . J. J. Pharm. Pharmacol. 1974, 26, 115P-116. Haworth, E.; Macdonald, A. D. J. Hyg. 1937, 3 7 , 234-42. N i c h o l l s , P. J. Br.J. Ind. Med. 1962, 19, 33-41. Northrup, S . ; Presant, L.; Kilburn, K. H.; McCormick, J.; Pachlatko, P . Fed. Proc, F e d . Am. Soc. Expt. Biol. 1976, 35, P632. Rylander, R., Mattsby, I., Snella, M-C. Chest 1979, 75S, 278S-9S. Furness, G.; Maitland, Η. B. Br. J. Ind. Med. 1952, 9, 138-45. Cavagna, G.; Foa, V.; Vigliani, C . Br. J. Ind. Med. 1969, 26, 314-21. Pernis, B.; Vigliani, C.; Cavagna, C.; Fenulli, M. B r . J . Ind. Med. 1961, 18, 120-9. Massoud, Α.; Taylor, G . Lancet 1964, 2, 607-10. O e h l i n g , Α.; Gonzales de la Reguerra, I.; Vines Rueda, J. J. R e s p i r a t i o n 1972, 2 9 , 155-60.


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28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.

DUST

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RECEIVED

January

20,

1982.


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