Activation of Leukocytes by ArgâGlyâAspâSer-Carrying

Chapter 19

Polymers of Biological and Biomedical Significance Downloaded from pubs.acs.org by NATL UNIV OF SINGAPORE on 11/10/17. For personal use only.

Activation of Leukocytes by Arg—Gly—Asp—SerCarrying Microspheres 1

1

2

1

Keyi Fujimoto , Y. Kasuya , M. Miyamoto , and H. Kawaguchi 1

Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223, Japan Department of Research, Central Blood Center, Japanese Red Cross, 4 Hiroo, Shibuya, Tokyo 150, Japan

2

It is known that a tetrapeptide, Arg-Gly-Asp-Ser (RGDS) has cell adhesion activity and plays an important role in cell-cell and cell-extracellular matrix interactions. RGDS-carrying microspheres, RGES-carrying microspheres, and parent microspheres were prepared to elucidate the RGDS-mediated activation of leukocytes. The ingestion of microspheres by leukocytes, the rate of oxygen consumption, the production of superoxide anion and hypochlorite ion and the inhibition of these products were examined. There was no difference in the number of microspheres ingested by leukocytes among the three kinds of microspheres. Leukocytes appeared specific to have an increase in oxygen consumption when they were mixed with RGDS-carrying microspheres. Moreover, this specificity was lowered by adding soluble RGDS or RGES-carrying microspheres. These findings indicate that the specific increase in oxygen consumption is caused by a signal transmission induced by immobilized RGD-integrin binding. Furthermore, it was found that the profiles of liberation of superoxide anion were similar to those of oxygen consumption. It seems probable that leukocytes are activated to produce active oxygens by interaction with RGDS peptides. In addition, specific oxygen consumption was inhibited by inhibitors of the growth of the actin filament. Immobilized RGDS may adhere to integrins and lead to the clustering of integrins and a change in the actin filament, and consequently cells may recognize the signal of activation through the actin filament. These results suggest that the microsphere with immobilized RGDS can be utilized as an activator of neutrophils. Microspheres containing immobilized bioactive molecules are used not only in cell targetting and separation, but in cell activation as well,


19. FUJIMOTO ET AL.

Activation of Leukocytes by Microspheres

221

b y t a k i n g a d v a n t a g e of t h e i r b i o s p e c i f i c affinity. In t h i s w o r k , we f o c u s e d o n a t e t r a p e p t i d e , A r g - G l y - A s p - S e r (RGDS), w h i c h is well k n o w n to be l o c a t e d w i t h i n f i b r o n e c t i n a n d is a b l e to p a r t i a l l y s u b s t i t u t e its function(i). The a d h e s i v e i n t e r a c t i o n of cells w i t h e x t r a c e l l u l a r matrix components s u c h as f i b r o n e c t i n p l a y s a n important role i n not o n l y cell a d h e s i o n b u t a l s o m a n y o t h e r c e l l u l a r f u n c t i o n s , i.e.: c e l l m o r p h o l o g y , g r o w t h , differentiation, a n d proliferation(J?). A s p r e v i o u s l y reported, immobilized RGDS p l a y e d a role i n mediating the a d h e r e n c e a n d a c t i v a t i o n o f p l a t e l e t s ( K a s u y a , Y . e t a l . J. Biomater. ScL Polymer Edn, in press.). L e u k o c y t e s also express a g l y c o p r o t e i n , " i n t e g r i n " , as a cell surface adhesion receptor. This g l y c o p r o t e i n recognizes the RGDS s e q u e n c e ^ ) a n d affects the l e u k o c y t e ' s functions(4,5). Yet little is k n o w n about the molecular events. I n t h i s w o r k , t h e r e f o r e , we t r i e d to p r e p a r e RGDS t e t r a p e p t i d e s a n d immobilize them on the p o l y m e r i c m i c r o s p h e r e s a n d i n t e n d e d to i n v e s t i g a t e the R G D S - m e d i a t e d a c t i v a t i o n of l e u k o c y t e s u s i n g RGDS-carrying microspheres. Experimental P r e p a r a t i o n of R G D S - c a r r y i n g m i c r o s p h e r e s Z-Arg(Mbs)-Gly-Asp(OBz)-Ser(Bzl)-OH (protected RGDS-OH) was synthesized by solution method(6). Substrate microspheres were p r e p a r e d b y soap free emulsion c o p o l y m e r i z a t i o n of s t y r e n e , a c r y l a m i d e and divinylbenzene. RGDS-carrying microspheres were prepared in a m a n n e r d e p i c t e d i n F i g u r e 1. Amino g r o u p s were p r o d u c e d on the substrate microspheres b y c a r r y i n g out the Hofmann reaction (parent m i c r o s p h e r e s ) a n d p r o t e c t e d RGDS was c o u p l e d onto the amino g r o u p s with carbodiimide, followed b y deprotection. As a control, R G E S - c a r r y i n g m i c r o s p h e r e s , w h i c h have no cell a d h e s i o n a c t i v i t y , w e r e p r e p a r e d . The amount of peptide immobilized on the m i c r o s p h e r e s was d e t e r m i n e d from the amino acid a n a l y s i s of the s u p e r n a t a n t after h y d r o l y s i s of the peptide b o n d s . G l y c y l g l y c i n e (GG) w a s u s e d as a s p a c e r . E v a l u a t i o n of p h a g o c y t o s i s P o l y m o r p h o n u c l e a r l e u k o c y t e s were isolated from f r e s h human blood b y t h e D e x t r a n - F i c o l l m e t h o d a t 25 °C. T h e y w e r e s u s p e n d e d i n p h o s p h a t e buffered s a l i n e ( p H 7.4) a t 10 cells m l . Two milliliters of this s u s p e n s i o n w a s m i x e d w i t h 100 μ. I of m i c r o s p h e r e d i s p e r s i o n . The interaction between leukocytes and various microspheres was first evaluated by measuring oxygen consumption. The c o n c e n t r a t i o n of o x y g e n i n the media was determined with" an o x y g e n electrode. Soluble RGDS a n d RGES (Bachem F i n e C h e m i c a l s Inc., U S A ) w e r e u s e d to examine the competitive reaction between soluble peptide a n d immobilized one. Colchicine and cytochalasin D (Cyt.D) (Sigma Chemical Co. L t d . , U S A ) were u s e d to i n h i b i t the s i g n a l t r a n s m i s s i o n i n d u c e d b y I n t e g r i n . T h i n c r o s s - s e c t i o n s of microspheres i n t e r n a l i z e d b y a l e u k o c y t e were observed with transmission electron microscopy (TEM). 7

- 1

E v a l u a t i o n of a c t i v a t i o n of l e u k o c y t e s The l u c i f e r i n - and the luminol-enhanced chemiluminescence were m e a s u r e d b y a m u l t i c h a n n e l l u m i n e s c e n c e a n a l y z e r ( B I O L U M A T L B 9505, B e r t h o l d , Wilbad, G e r m a n y ) i n o r d e r to d e t e r m i n e a c t i v e o x y g e n s l i b e r a t e d from l e u k o c y t e s . L u c i f e r i n d e r i v a t i v e a n d l u m i n o l are r e a g e n t s u n i q u e to s u p e r o x i d e a n i o n ( 0 ~ ) a n d h y p o c h l o r i t e i o n (0C1~), r e s p e c t i v e l y . 2


222

POLYMERS OF BIOLOGICAL AND BIOMEDICAL SIGNIFICANCE

St + A A m + DVB

ι Ο 1

soap-free emulsion copolymerization

CONHa Hofmann reaction

Ο

parent microsphere

protected RGDS-OH ,(RGES) DIPCDI, HOBt

deprotectîon

Boc-GG-OH

ψ RGDS-NH-

Boc-GG \

)

protected RGDS-OH -τ

, (RGES) '

RGDS-GG-NH

Figure 1. Preparation of RGDS-carrying microspheres. St: styrene, A A m : acrylamide, DVB: divinylbenzene, DIPCDI: diisopropylcarbodiimide, HOBt: 1-hydroxybenzotriazole.

19. FUJIMOTO ET AL.

Activation of Leukocytes by Microspheres 223

Results & Discussion The microspheres obtained b y soap-free emulsion polymerization w e r e m o n o d i s p e r s e a n d 294 nm i n d i a m e t e r . T h e a m o u n t o f a m i n o g r o u p s o n t h e a m i n o m i c r o s p h e r e s w a s 4.3 ± 0 . 4 u n i t s n m . T h e a m o u n t o f i m mobilized peptide a n d the zeta potentials of the microspheres are s h o w n i n T a b l e 1. F i g u r e 2 shows the o x y g e n consumption b y leukocytes phagocytosing parent microspheres and RGDS- and RGES-carrying m i c r o s p h e r e s as a f u n c t i o n of i n c u b a t i o n time. A t the b e g i n n i n g of i n cubation, both RGDSand RGES-carrying microspheres were p h a g o c y t o s e d to a l e s s e r extent t h a n p a r e n t ones. This is p r o b a b l y because immobilization of peptides r e n d e r s microspheres h y d r o p h i l i c , independent of the affinity of peptides. In the RGES-carrying microsphere system, oxygen consumption proceeded slowly and remained stable at a low l e v e l . On the o t h e r h a n d , l e u k o c y t e s c o n s u m e d o x y g e n v e r y u n i q u e l y when they were mixed with R G D S - c a r r y i n g microspheres. P h a g o c y t o s i s was a c c e l e r a t e d at a r o u n d 5 min, g r a d u a l l y i n c l i n e d a n d l e v e l e d o f f a f t e r 20 m i n ( s e c o n d s t a g e ) . W h e n o n l y s o l u b l e R G D S w a s a d d e d to l e u k o c y t e s , o x y g e n c o n s u m p t i o n c o u l d not be d e t e c t e d . These results indicate that RGDS-mediated phagocytosis takes place with a s h o r t lag a n d immobilization of RGDS leads to u n i q u e o x y g e n c o n s u m p t i o n of l e u k o c y t e s . M o r e o v e r , it c a n be o b s e r v e d from F i g u r e 2 t h a t o x y g e n c o n s u m p t i o n at the s e c o n d stage was s u p p r e s s e d b y d o s e s of free RGDS. Evidently, this unique o x y g e n c o n s u m p t i o n is associated w i t h the s p e c i f i c i t y of the immobilized RGDS for i n t e g r i n ( V L A - 5 ) of l e u k o c y t e s .


- 2

F u r t h e r m o r e , it was o b s e r v e d from F i g u r e 2 that the i n t r o d u c t i o n of a s p a c e r GG a c c e l e r a t e d p h a g o c y t o s i s of R G D S - c a r r y i n g m i c r o s p h e r e s b y a f a c t o r o f a b o u t 1.3. T h i s i n d i c a t e s t h a t g r e a t e r f l e x i b i l i t y i n o r i e n t a t i o n of the RGDS p e p t i d e a l l o w s b e t t e r r e c o g n i t i o n . B u t t h e r e is no d e n y i n g the p o s s i b i l i t y t h a t GG a l t e r e d the c o n f o r m a t i o n of RGDS so as to i n c r e a s e the affinity for integrin. T r a n s m i s s i o n e l e c t r o n m i c r o s c o p i c o b s e r v a t i o n was p e r f o r m e d to i n vestigate the relation between the ingestion and the unique o x y g e n c o n s u m p t i o n . T h e r e was no d i f f e r e n c e i n the n u m b e r of m i c r o s p h e r e s i n g e s t e d b y l e u k o c y t e s among the t h r e e k i n d s of m i c r o s p h e r e s . This f i n d i n g s t r o n g l y s u g g e s t s that the u n i q u e o x y g e n c o n s u m p t i o n is not r e l a t e d to i n g e s t i o n b u t c a u s e d b y t h e s i g n a l t r a n s m i s s i o n i n d u c e d b y immobilized RGDS-integrin b i n d i n g . P r o d u c t i o n of active o x y g e n s was measured b y the l u c i f e r i n - a n d the l u m i n o l - e n h a n c e d chemiluminescence to make c l e a r the c a u s e of o x y g e n c o n s u m p t i o n a t t h e s e c o n d s t a g e . A s c a n b e s e e n i n F i g u r e 3, t h e p r o f i l e s of l i b e r a t i o n of s u p e r o x i d e a n i o n w e r e s i m i l a r to t h o s e of o x y g e n consumption. T h i s s u g g e s t s t h a t l e u k o c y t e s might be a c t i v a t e d by immobilized RGDS to p r o d u c e a c t i v e o x y g e n s . On the o t h e r h a n d , the luminol-dependent chemiluminescence revealed little difference i n the time-course of active oxygen production among three kinds of m i c r o s p h e r e s a s s h o w n i n F i g u r e 4. Probably, this is because the f o r m a t i o n of 0C1~ m i g h t be m u c h more s t r o n g l y a f f e c t e d b y v a r i o u s enzymes released from l e u k o c y t e s t h a n b y the extent of a c t i v a t i o n v i a RGDS-integrin binding. The effect of i n h i b i t o r s of cytoskeletons on the total amount of


224


T a b l e 1. T h e a m o u n t o f p e p t i d e i m m o b i l i z e d o n t h e a n d zeta p o t e n t i a l s of the m i c r o s p h e r e s immobilized

chain

immobilized (unit nm~ ) 2

parent microspheres RGDS RGES GG RGDS-GG RGES-GG

4.210.4 2.610.2 5.110.5 4.610.2 2.910.1

peptide

microspheres

zeta potential (mV) 13.1 1.7 14.4 13.8 .1.4 13.2

Figure 2. Phagocytosis of peptide-carrying microspheres by leukocytes, (a) parent, (b) RGDS-GG-carrying, (c) RGDS-carrying, (d) RGES-carrying, (e) RGES-GG-carrying microspheres, and (f) soluble RGDS.


19. FUJIMOTO ET AL.


225

Incubation tlme/min

Figure 3. Time course of luciferin-dependent chemiluminescence of leukocytes stimulated by microspheres, (a) parent, (b) RGDS-GG-carrying, (c) RGESGG-carrying microspheres and (d) absence of microspheres.

30 Incubation time/min

Figure 4. Time course of luminol-dependent chemiluminescence of leukocytes stimulated by microspheres, (a) parent, (b) RGDS-GG-carrying, (c) RGESGG-carrying microspheres, and (d) absence of microspheres.


226


Figure 5. Effect of inhibitors on oxygen consumption of leukocytes induced by microspheres (Cyt.D: 100 #g mT" , colchicine: 500 μΜ). Error bars show standard deviations.


19. FUJIMOTO ET AL.


227

o x y g e n c o n s u m p t i o n i s s h o w n i n F i g u r e 5. C y t . D b i n d s t o t h e a c t i n filament, r e s u l t i n g i n the i n h i b i t i o n of the g r o w t h of the a c t i n filament. Figure 5 shows that oxygen consumption in the RGDS-carrying m i c r o s p h e r e s y s t e m seemed to be more e f f e c t i v e l y i n h i b i t e d b y C y t . D t h a n t h a t of the o t h e r s . H o w e v e r , t h e r e was no s i g n i f i c a n t d i f f e r e n c e i n the r e d u c t i o n of o x y g e n c o n s u m p t i o n among three k i n d s of microspheres when leukocytes were pretreated b y colchicine, which inhibits the p o l y m e r i z a t i o n of m i c r o t u b u l e s . M o r e o v e r , it c o u l d be o b s e r v e d t h a t the second stage of R G D S - c a r r y i n g microspheres d i s a p p e a r e d b y the a d d i t i o n of C y t . D . It i s w e l l k n o w n t h a t t h e i n t r a c e l l u l a r d o m a i n s o f i n t e g r i n s i n t e r a c t w i t h p r o t e i n s s u c h as talin, v i n c u l i n , a n d a - a c t i n i n . These p r o t e i n s link a n i n t e g r i n to the a c t i n filaments. T h e r e f o r e , it seems that immobilization of RGDS leads to a c h a n g e i n the a c t i n filament a n d t h a t t h i s c h a n g e is r e l a t e d to c l u s t e r i n g of i n t e g r i n s . We i m a g i n e t h a t s p e c i f i c a c t i v a t i o n o f c e l l s r e q u i r e s t h e a f f i n i t y a n d the m i c r o e n v i r o n m e n t a c c o r d i n g to e a c h s i g n a l t r a n s d u c t i o n , because the signal is t r a n s d u c e d t h r o u g h the conformational changes a n d c l u s t e r i n g of r e c e p t o r s \ A s p o l y m e r i c m i c r o s p h e r e s a r e the same s i z e as c e l l s , t h e y are e x p e c t e d to be s u i t a b l e s u p p o r t s f o r the s i g n a l t r a n s d u c t i o n . In t h i s s t u d y , an increase i n a c t i v i t y was not seen i n the presence of soluble RGDS o r RGES-carrying microspheres. Moreover, the presence of m i c r o s p h e r e s alone o r the presence of m i c r o s p h e r e s a n d soluble RGDS d i d not p r o d u c e an increase i n a c t i v i t y . Only the microspheres with attached RGDS produced an increase in activity. These indicate that the mechanism of the b i o s p e c i f i c a c t i v a t i o n is s y n e r g i s t i c . P r o b a b l y , the m i c r o s p h e r e s w i t h i m m o b i l i z e d RGDS c a n a d h e r e to n e u t r o p h i l s v i a i n t e g r i n s a n d l e a d to a c h a n g e i n the a c t i n filament a n d to c l u s t e r i n g of i n t e g r i n s , a n d c o n s e q u e n t l y cells c a n r e c o g n i z e these c h a n g e s as the s i g nal of a c t i v a t i o n . 7

Literature Cited 1. 2. 3. 4.

Pierschbacher,M.D.; Ruoslahti,E. Nature, 1984, 309, 30. Ruoslahti,E. Ann. Rev. Biochem., 1988, 57, 375. Brown,E.J.; Goodwin,J. J. Exp. Med., 1988, 167, 777. Singer,I.I.; Scott,S.; Kawka,D.W.; Kazazis,D.M.; J. Cell Biol., 1989, 109, 3169. 5. Gresham,H.D.; Goodwin,J.; Allen,P.M.; Anderson,D.C.; Brown,E.J.; J. Cell Biol., 1989, 108, 1935. 6. Principles of Peptide Synthesis,; Bodanszky,M.Ed.; Springer-Verlag, Berlin, Heidelberg, 1984. 7. Kornberg,L.J.; Earp,H.S.; Turner,C.E.; Prockop,C.; Juliano,R.L.; Proc. Natl. Acad. Sci. USA, 1991, 88, 8392. RECEIVEDJuly6, 1993

Activation of Leukocytes by ArgâGlyâAspâSer-Carrying

Recommend Documents

Activation of Leukocytes by ArgâGlyâAspâSer-Carrying