Polymeric Materials and Artificial Organs - American Chemical Society

3 Artificial Organs and the Immune Response P. Y. W A N G and C. C H A M B E R S

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Laboratory of Chemical Biology, Institute of Biomedical Engineering & Department of Physiology, Faculty of Medicine, University of Toronto, Ontario, Canada M5S 1A8

Availability and immunological limitations of homografts have stimulated the development of artificial organs. Many extracorporeal and implantable devices have achieved remarkable success in substituting defective natural functions. Recently, interests in developing controlled release delivery systems to mimic the functions of the endocrines, e.g., in contraceptive steroid delivery, may extend this area of biotechnology to affect a large number of healthy individuals. Possible immune responses to synthetic materials used in fabrication should be among important aspects of artificial organ research. Antigenicity can be assessed by many antibody detection methods. The radioimmunoassay method is particularly suitable and our results show that C57BL/6 mice are high responders to many water-soluble biomedical polymers of acrylic acid, acrylamide, vinylpyrrolidone, vinyl alcohol, Na styrenesulfonate, etc. Others have observed that some of these polyanions can influence the outcome of the immune response. Insoluble polymers are not as easily assessed, but an indirect method has shown that several inert inorganic and organic biomedical polymers can also alter the immune response. The scanty information available at present already indicates that artificial organs may not be immune to responses from the immune system. +

Advances i n modern m e d i c a l p r a c t i c e have i n c r e a s e d the use p l a s t i c m a t e r i a l s f o r t i s s u e or organ replacements as w e l l

0097-6156/ 84/ 0256-0031 $06.00/0 © 1984 American Chemical Society

Gebelein; Polymeric Materials and Artificial Organs ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

of as


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POLYMERIC MATERIALS AND ARTIFICIAL ORGANS

i n devices which may help t o r e s t o r e the p h y s i o l o g i c a l s t a t e of the body. P r i o r t o use, the s u i t a b i l i t y o f a m a t e r i a l f o r biomedical a p p l i c a t i o n s must be e s t a b l i s h e d . Often, the m a t e r i a l performance i s assessed by measuring the p h y s i c a l , chemical, and b i o c o m p a t i b i l i t y p r o p e r t i e s . Since they do not occur i n nature, many s y n t h e t i c polymers are b e l i e v e d t o generate no immune response. There i s s u f f i c i e n t information a v a i l a b l e a t present to i n d i c a t e t h a t an immune response can indeed be induced by some polymer components i n drug formulations o r i n s u r g i c a l implants. This paper b r i e f l y surveys the immunological events from p r e s e n t l y r a t h e r l i m i t e d amount o f a v a i l a b l e data i n this developing area of b i o m a t e r i a l s research. H i s t o r i c a l Background (1) The e a r l y study o f the immune response evolved mostly from c l i n i c a l o b s e r v a t i o n of i n f e c t i o u s d i s e a s e s . The ''resistance" to a second i n f e c t i o n of the same disease was noted around 500 B.C. An e a r l y form of vaccine was the r a t h e r unsafe use o f l i v e smallpox organisms given t o people f o r p r o t e c t i o n against infection. L a t e r , Jenner v a c c i n a t e d against smallpox w i t h the n o n - v i r u l e n t cowpox organism. However, i t was not u n t i l the l a t e 19th century that the r o l e o f a n t i b o d i e s i n the defense against i n f e c t i o n became g r a d u a l l y apparent. Soon, the presence of a substance i n the serum t h a t could combine w i t h r e d blood c e l l s i n v i t r o and made the c e l l s r e s i s t a n t to the r i c i n t o x i n was observed by E h r l i c h . The o b s e r v a t i o n was the f i r s t evidence of antigen-antibody complexing which was part o f the defense mechanism. This l e d t o f u r t h e r s t u d i e s of antigen (abbrev. as Ag) immunochemistry, and the immunology of antibody (abbrev. as Ab) p r o d u c t i o n , p a r t i c u l a r l y w i t h p r o t e i n s , p l a n t and b a c t e r i a l products. With the d i s c o v e r y o f the ABO types i n human blood group substances by L a n s t e i n e r , the genetic c o n t r o l of the immune system was a l s o r e a l i z e d . Concepts o f the Immune System ( 1 , 2) The adaptive or acquired immune system i n mammals i s c h a r a c t e r i z e d by three f e a t u r e s , i . e . , s p e c i f i c i t y , r e c o g n i t i o n , and memory. These features depend on the i n t r i n s i c a b i l i t y o f the lymphocytes t o recognize a substance as f o r e i g n , and a l l o w an increased e f f i c i e n c y o f response upon a second exposure t o the same substance. An Ag i s a m a t e r i a l capable o f r e a c t i n g s p e c i f i c a l l y w i t h the Ab produced as a m a n i f e s t a t i o n o f the response. I t i s a l s o p o s s i b l e f o r an Ag, e s p e c i a l l y i n high doses, t o produce a s t a t e o f non-responsiveness r e f e r r e d t o as tolerance. For an Ag t o induce a response, i t should have a r e l a t i v e l y l a r g e molecular weight, e.g., g r e a t e r than 1,000



3.

WANG AND CHAMBERS

Artificial

Organs and the Immune Response

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d a l t o n s . Otherwise, i t i s r e f e r r e d t o as a hapten which would not induce a response by i t s e l f , unless l i n k e d t o a polymeric " c a r r i e r " such as p o l y - ( v i n y l a l c o h o l ) , p o l y - acrylamide, etc. The immune response produced i s a f f e c t e d by the p r e s e n t a t i o n o f the Ag t o the host, e.g., the s t a t e o f the Ag, adjuvant, dose, r o u t e , e t c . , as w e l l as by genetic f a c t o r s . The primary e f f e c t o r s o f the immune system are the lymphocytes, and t h e i r precursors are the stem c e l l s i n the bone marrow. One sub-population o f the stem c e l l s d i f f e r e n t i a t e s i n the thymus ( i . e . the T - c e l l s ) , w h i l e the other matures p r i m a r i l y i n the bone marrow and the g u t - a s s o c i a t e d lymphoid t i s s u e s ( i . e . the B - c e l l s ) . A c c o r d i n g l y , there are two types o f responses depending on the c e l l sub-population that i s a c t i v a t e d . The f i r s t i s the c e l l mediated response, i f the T-lymphocytes a r e stimulated. The other i s the humoral response, i f only t h e B - c e l l s are i n v o l v e d . However, most B - c e l l s which respond by r e l e a s i n g Ab a l s o may r e q u i r e a T - e e l l " h e l p e r " s i g n a l t o transform and p r o l i f o r a t e i n t o plasma c e l l s . This s e r i e s o f events i s f u r t h e r complicated by the r e g u l a t i o n o f such responses which i n v o l v e T-suppressor c e l l s , p a r t i c i p a t i o n o f macrophages, other accessory c e l l s , negative feedback from the Ab themselves, genetic c o n t r o l s , and p o s s i b l y other f a c t o r s yet to be d e l i n e a t e d . Antibody D e t e c t i o n Methods (3) There are many w e l l - e s t a b l i s h e d methods f o r the d e t e c t i o n o f immune responses, e s p e c i a l l y when the Ab are r e l e a s e d i n t o the systemic c i r c u l a t i o n . E a r l i e r , the a g g l u t i n a t i o n o f b a c t e r i a was used t o show the presence o f s p e c i f i c Ab i n serum o f infected individuals. The formation o f Ag-Ab complex can a l s o be observed as aggregates i n the r i n g t e s t , p r e c i p i t i n r e a c t i o n or v a r i o u s g e l media. More r e c e n t l y , s e n s i t i v e methods a r e developed t o d e t e c t Ab i n the ug t o pg range. D e t a i l s o f the hemagglutination, hemolysis, radioimmunoassay (abbrev. as RIA) and plaque forming assay, e t c . , can be found i n a standard manual ( 3 ) . The l i m i t s o f s e n s i t i v i t y o f these methods a r e shown i n Table I . However, a l l these Ab analyses are devised to study n a t u r a l o r haptenic Ag, and may not always be a p p l i c a b l e t o the d e t e c t i o n o f Ag-Ab i n t e r a c t i o n i n v o l v i n g h i g h l y w a t e r - s o l u b l e n o n - b i o l o g i c a l polymers t h a t are strong polyelectrolytes. Thus, adaption and use o f these c l a s s i c a l methods to the s y n t h e t i c polymers should be made w i t h c a u t i o n . Experimental S e v e r a l mouse s t r a i n s were observed t o produce Ab a f t e r s t i m u l a t i o n w i t h water-soluble s y n t h e t i c polymers ( 4 ) . The C57BL/6 s t r a i n was found t o be most responsive. Therefore, i n t h i s study s e v e r a l common w a t e r - s o l u b l e polymer Ag were used t o


34


Table I . A n a l y t i c a l S e n s i t i v i t y o f Some D e t e c t i o n Methods f o r Ab


Methods Precipitation Gel D i f f u s i o n Hemagglutination Hemolysis Complement F i x a t i o n RIA

Sensitivity (UK Ab/ml serum) 20 3 t o 200 0.001 t o 1 0.0001 t o 0.01 0.1 0.001

induce Ab production i n groups of f i v e C57BL/6 mice f o r each experiment. The polymer Ag were e m u l s i f i e d i n Freund's complete adjuvant, and i n j e c t e d i n t r a p e r i t o n e a l l y as d e s c r i b e d p r e v i o u s l y ( 4 ) . A t r e g u l a r i n t e r v a l s , the rodents were b l e d and the sera were separated from the coagulated c e l l u l a r components ( 4 ) . Presence of Ab i n the sera was detected by the s o l i d phase RIA. B r i e f l y , 50 u l a l i q u o t s o f a s p e c i f i c Ag s o l u t i o n c o n t a i n i n g 2% bovine serum albumin (abbrev. as BSA) were added t o a number o f the 96-well p l a s t i c assay p l a t e which was then incubated f o r 2 hr a t 37°C t o a l l o w the Ag t o become adsorbed onto the p l a s t i c . The Ag-coated w e l l s were washed 3x w i t h a BSA~containing b u f f e r , and 50 μΐ a l i q u o t s o f a s e r i a l l y d i l u t e d Ab s o l u t i o n were added a c c o r d i n g l y t o the coated wells. After 2 hr a t room temperature f o r Ab i n t e r a c t i o n w i t h the adsorbed Ag, the w e l l s were again washed, and then t r e a t e d w i t h 50 y l a l i q u o t s of an " ^ I - l a b e l l e d r a b b i t anti-mouse Ab fragment. The p l a t e was incubated a t 4°C f o r 16 h r , and the w e l l s were washed once more. A f t e r c u t t i n g the w e l l s a p a r t , the l e v e l s o f gamma-ray emitted by each sample were counted, and the r e s u l t s were reported as the maximum i n geometric s e r i a l d i l u t i o n s o f the anti-serum that showed Ab a c t i v i t y a f t e r deduction of any background a c t i v i t i e s from the c o n t r o l which had normal mouse serum i n place o f the Ab solution. Results S e v e r a l r e a d i l y a v a i l a b l e biomedical polymers (Table I I ) were used as Ag t o induce immune response i n C57BL/6 mice and the Ab i n the s e r a were detected by RIA. The r e s u l t s i n Table I I show that under the present experimental c o n d i t i o n s , PVA, PAA, and


3.

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WANG AND CHAMBERS


35

Ρ AM are weakly o r moderately immunogenic, w h i l e PSS and PVP are more potent Ag comparable t o some n a t u r a l Ag. T y p i c a l l y , the more immunogenic PSS e x h i b i t s a dose response r e l a t i o n s h i p (Table I I I ) . Doses as low as 1 0 ~ yg can induce d e t e c t a b l e Ab by the RIA, but almost a m i l l i o n - f o l d increase i n the immunizing dose o f PSS i s r e q u i r e d i n order t o o b t a i n a substantially higher Ab t i t r e , and t h e o p t i m a l response decreases r a t h e r r a p i d l y (Table I I I ) . An apparent secondary response i s observed when the mice were immunized w i t h a sub-optimal dose f o l l o w e d by a booster i n j e c t i o n (Table I V ) . This secondary response decreased t o a lower l e v e l of Ab t i t r e i n about 1 week. S i m i l a r l y , PVP i s a l s o known t o induce such responses i n mice ( 5 ) .


3

Table I I . A n t i g e n i c Biomedical Polymers Tested i n C57BL/6 Mice

Abbrev. Names

Max. RIA Ab T i t r e (2 dilutions)

a

n

PSS PVP PVA PAA PAM

a

4096 4096 64 1024 16

b

+

PSS: P o l y ( s t y r e n e s u l f o n a t e Na ) ; PVP: P o l y v i n y l p y r r o l i done); PVA: p o l y v i n y l a l c o h o l ) ; PAA: P o l y U c r y l i c a c i d ) ; PAM: P o l y ( a c r y l a m i d e ) . ^Detected by hemagglutination assay; immune serum from SJL mice.

Table I I I .

Dose Response R e l a t i o n s h i p f o r PSS i n C57BL/6 Mice

Dose (yg) '"~{^~3

10~ 1010 5 χ 10 8 χ 10 10 5 χ 10

2

1

2

2 2

3

3

Max. RIA Ab T i r e (2 dilutions) n

256

256 256 256 1024 512 4096 512


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Table IV.

Responses t o Two I n j e c t i o n s o f 0.1 yg PSS i n C57BL/6 Mice

RIA Ab T i t r e (2 dilutions) n


Days 0 (1st I n j . ) 10 15 28 35 38 (2nd I n j . ) 42 50 58 66

128 256 32 16 1024 256 256 16

Discussion In our previous r e p o r t (4, !5 ), the a n t i g e n i c i t y o f some o f the biomedical polymers shown i n Table I I has been evaluated by using hemagglutination as the Ab d e t e c t i o n method. However, probably because o f the chemical or p h y s i c a l c h a r a c t e r i s t i c s o f these s y n t h e t i c polymers, t h i s method does not always produce predictable results. The s o l i d phase RIA has c o r r e c t e d t h i s i n c o n s i s t e n c y , and most o f the Ab t i t r e s shown i n Table I I t o IV are r e p r o d u c i b l e t o w i t h i n 2 - d i l u t i o n s which are i n the range o f v a r i a t i o n f o r t h i s type o f experiments. Therefore, s y n t h e t i c polymers, some as shown i n Table I I a t l e a s t , are not n e c e s s a r i l y i n a c t i v e t o the immune system. Although there are s u b s t a n t i a l amount of data a v a i l a b l e on responses t o s y n t h e t i c poly(amino a c i d s ) which are e s s e n t i a l l y pseudo-biological polymers, a survey o f l i t e r a t u r e s has provided i n f o r m a t i o n mostly on PVP. For example, Andersson and coworkers (6) have shown that PVP can induce Ab production i n adult thymectomized mice after irradiation, f o l l o w e d by r e c o n s t i t u t i o n w i t h B - c e l l s i s o l a t e d from the spleens o f other non-treated mice w i t h the same genetic background. Their experiment demonstrates that PVP i s a T-independent Ag ( 4 ) . Further s t u d i e s have shown that such response i n r e c o n s t i t u t e d mice i s a f f e c t e d by the m a t u r i t y of the B - c e l l s , and i n younger animals, the co-operation o f T - c e l l s i s r e q u i r e d (7.). I n a d d i t i o n , others have observed t h a t low doses o f PVP may s t i m u l a t e a sub-population o f T - c e l l s (the helper c e l l s ) t o n

2



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Artificial


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enhance the Ab production by B - c e l l s ( 8 ) . The opposite e f f e c t occurs when higher o r the optimal dose i s given ( 9 ) . There are a l s o r e p o r t s i n d i c a t i n g t h a t p o l y ( a c r y l i c acid) can be used t o r e p l a c e the h e l p e r c e l l f u n c t i o n s aforementioned i n the response t o the T-dependent 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 (10). For many w a t e r - i n s o l u b l e polymers the immune response, i f any, i s more d i f f i c u l t t o evaluate. Recently, Habal and coworkers (11) have reported a method t o assess the e f f e c t of s o l i d implant m a t e r i a l s such as s i l i c o n e , segmented p o l y e t h e r polyurethane, poly(methyl m e t h a c r y l a t e ) , and B i o g l a s using tumor-bearing mice as the experimental model. They have found that the B - c e l l s from the t e s t animals showed a reduced c a p a c i t y f o r p r o l i f e r a t i o n when s t i m u l a t e d by mitogens as compared t o the c o n t r o l s . The r e s u l t s demonstrate once again that even the r e l a t i v e l y biocompatible s o l i d polymers may have a measurable e f f e c t on the immune system. Besides the many immunological events that can be i n i t i a t e d by the s y n t h e t i c polymers as j u s t d e s c r i b e d , there are a l s o the p a t h o l o g i c a l i m p l i c a t i o n s t o be considered, such as the i n v i v o f a t e o f the Ag-Ab complexes. Consequently, there i s an urgent need f o r emphasis on immunological s t u d i e s o f b i o m a t e r i a l s . Meanwhile, from our data and the i n f o r m a t i o n i n the l i t e r a t u r e as j u s t d e s c r i b e d , i t appears that a r t i f i c i a l organs may not be immune t o immune responses. But the long-term e f f e c t s o f such responses as w e l l as these e f f e c t s on the complement system seem to be much more complex than are r e a l i z e d a t present. Literature Cited 1. R o i t t , I . "Essential Immunology"; Blackwell S c i e n t i f i c : London, 1980; p. 4. 2. Raff, M.C. S c i e n t i f . Amer. 1976, 234, 30. 3. M i s h e l l , B . B . ; S h i i g i , S.M. "Selected Methods in C e l l u l a r Immunology"; W.H. Freeman: San Francisco, 1980. 4. Wang, P.Y. Advan. Biomaterials 1982, 3, 799. 5. Wang. P . Y . ; Alouf, Α.; Samji, N . ; Wolinsky, S. Immunology '80 (4th I n t e r n a t ' l . Congr. Immunology, I n t e r n a t ' l . Union Immunol.) 17.4.21. 6. Andersson, B . ; Blomgren, H. C e l l . Immunol. 1971, 2, 411. 7. Andersson, B . ; Blomgren. H. Nature 1975, 253, 476. 8. Braley-Mullen, H . ; L i t e , H. Develop. Immunol. 1981, 15, 401. 9. Inaba, K . ; Nakano, K . ; Muramatsu, S. C e l l . Immunol. 1978, 39, 260. 10. Sjoberg, O.; Andersson, J.; Moller, G. Eur. J . Immunol. 1972, 2, 326. 11. Habal, M . B . ; Powell, M . L . ; Schimpff, R.D. J . Biomed. Mater. Res. 1980, 14, 455. RECEIVED March 19, 1984


Polymeric Materials and Artificial Organs - American Chemical Society

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