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IMMUNOPHARMACOLOGY A N D IMMUNOTOXICOLOGY, 1 3 ( 4 ) , 607-622 (1991)
INHIBITOTY EFFECTS OF LOCAL ANESTHETICS ON MIGRATION, EXTRACELLULAR RELEASE OF LYSOSOMAL ENZYME, AND SUPEROXIDE ANION PRODUCTION IN HUMAN POLYMORPHONUCLEAR LEUKOCYTES
Q
Sumiko Sasagawa Department of Pathology, Radiation Effects Research Foundation 5-2 Hijiyama Park, Minimi-ku, Hiroshima 7 3 2 , Japan.
ABSTRACT
This study examined the effects of four typical local anesthetics,lidocaine, prilocaine, procaine and tetracaine, on the functioning of human polymorphonuclear leukocytes (PMN). PMN were stimulated by fMet-Leu-Phe (FMLP) or phorbol myristate acetate (PMA) to elicit chemotaxis, extracellular release of betaglucuronidase (BGL) and superoxide anion (SOA) production. The four agents inhibited chemotaxis efficiently and in a concentration-dependent manner but had only weak effects on the release of BGL. The effect of tetracaine was strongest, followed by lidocaine, then prilocaine, whereas the effect of procaine was blunt. The 50% inhibitory concentrations (ICsO in molarity) of the four local a esthetics for chemetaxis were as follows: tetracaine prilocaine = 3.6 x 10- 3 , = 4 . 1 x 10- , lidoca’ne = 3.2 x those for SOA production induced by FMLP procaine = 4 . 9 x lo-’, were : tetra aine = 3.1 x lidoc ine = 5.9 x prilocaine procaine = 1.2 x lo-’, those for SOA production = 1.9 x ind ced by PMA were : tetracaine = 1.1 x 10- 3 , lidoc ine = 1.2 x 10- ! , prilocaine = 1.5 x 10- 2 , procaine = 2.5 5 10- , and those for r lease of BGL were : tetracaine = 1.6 x 10- , lidocaine = 5.3 x 10- 3 , prilocaine = 2.8 x 10- 2 , procaine = 1 . 2 x 10- 1. The IC50 seemed to relate to the anesthetic’s chemical structures and their inhibitory properties on P M N functions, a s lidocaine and prilocaine, which are aminoamide type anesthetics, preferentially inhibited chemotaxis, whereas tetracaine and procaine, aminoester type anesthetics, inhibited SOA production induced by FMLP. The results suggest that the inhibitory effects of local anesthetics on human PMN functions are also correlated with local anesthetic potency and vary according to differences in their chemical structures,
t
9
6C7 Copyright 0 1991 by Marcel Dekker, Inc.
SASAGAWA
608
INTRODUCTION ( 1 ) a r e n o n s p e c i f i c membrane-stabilizing
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Flinders University of South Australia on 01/12/15 For personal use only.
Local a n e s t h e t i c s s u b s t a n c e s which
affect
lipid
polymorphism,
c a u s e membrane
expansion, d i s p l a c e calcium i o n from b i n d i n g s i t e s , and depress facilitated
f l u x e s of
s o l u t e s a c r o s s c e l l membranes. The major
property of l o c a l a n e s t h e t i c s -- conduction blockade i n nerves --
i s r e f l e c t e d b y t h e i r chemical s t r u c t u r e . the
anesthetics
physicochemical
produce
properties,
Chemical a l t e r a t i o n s of
quantitative
i.e.,
lipid
changes
their
i n
s o l u b i l i t y and
protein-
b i n d i n g , w h i c h , i n t u r n , a l t e r t h e a n e s t h e t i c p r o f i l e of t h e compounds ( 2 ) . Local a n e s t h e t i c s a c t on many immunocompetent c e l l s a s w e l l a s on t h e nerve c e l l s . transformation ( 3 ) .
They i n h i b i t mitogen-induced
lymphocyte
I n v i v o ( 4 ) a n d i n v i t r o ( 5 ) e x p o s u r e of
mouse s p l e e n lymphocytes t o l i d o c a i n e r e s u l t e d i n changes i n t h e i r s u r f a c e c h a r g e p r o f i l e s and i n h i b i t i o n response t o mitogens.
L i d o c a i n e and
of
blastogenesis
p r o c a i n e both
inhibit
in the
r e c o g n i t i o n and t h e p o s t r e c o g n i t i o n p r o c e s s e s ( 6 ) and c e l l lysis
( 7 ) of human n a t u r a l
I t h a s a l s o been d e m o n s t r a t e d t h a t l o c a l
killer cells.
a n e s t h e t i c s have m u l t i p l e e f f e c t s on polymorphonuclear l e u k o c y t e s (PMN) ( 8 - l l ) , i . e . , production,
i n h i b i t i o n of
superoxide anion (SOA)
lysosomal enzyme r e l e a s e and a d h e s i o n .
c r u c i a l r o l e i n h o s t d e f e n s e mechanisms and
PMN play a
a l s o cause
tissue
damage i n t h e inflammatory s i t e owing t o t h e i r a b i l i t y t o m i g r a t e toward i n j u r e d and i n f e c t e d s i t e s , t o phagocytize microorganisms
in
a c t i v a t i o n of
their
o x i d a t i v e metabolism and r e l e a s e of lysosomal enzymes ( 1 2 ) .
It is
and t o k i l l
v a r i o u s ways
involving
expected t h a t l o c a l a n e s t h e t i c s i n h i b i t PMN f u n c t i o n s i n t h e same way a s conduction blockade i n n e r v e s and t h a t t h e r e i s a r e l a t i o n between t h e i r e f f e c t on PMN and t h e i r a n e s t h e t i c potency, s i n c e b o t h t y p e s of c e l l membranes d e p e n d e n t l y r e s p o n d t o
surface
s t i m u l i , a l t h o u g h t h e i r f u n c t i o n s i.n t h e body a r e d i f f e r e n t .
609
ANESTHETICS IN HUMAN POLYMORPHONUCLEAR LEUKOCYTE5
I n t h e p r e s e n t s t u d y , we t e s t e d f o u r t y p i c a l , r o u t i n e l y a v a i l a b l e l o c a l a n e s t h e t i c s , l i d o c a i n e and p r i l o c a i n e , which a r e aminoamides, and p r o c a i n e and t e t r a c a i n e , which a r e a m i n o e s t e r s , Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Flinders University of South Australia on 01/12/15 For personal use only.
and compared t h e potency of t h e i r a b i l i t y t o i n t e r f e r e w i t h t h e m i g r a t i o n , lysosomal enzyme r e l e a s e , and SOA production of human
PMN i n
response
to
s y n t h e t i c chemotactic
peptide
(FMLP) and phorbol m y r i s t a t e a c e t a t e (PMA) i n v i t r o .
fMet-Leu-Phe
The results
i n d i c a t e t h a t t h e i n h i b i t o r y e f f e c t of l o c a l a n e s t h e t i c s on PMN function is correlated w i t h
t h e i r a n e s t h e t i c potency.
The
d i f f e r e n c e s i n i n h i b i t o r y a c t i o n seen i n t h i s s t u d y a r e l i k e l y due t o t h e mechanisms of t h e a n e s t h e t i c s '
a c t i o n a s well a s t o t h e
mechanisms of PMN f u n c t i o n a t t h e inflammatory s i t e .
MATERIALS AND METHODS Materials :
FMLP was purchased from P r o t e i n Research Foundation
(Osaka), c y t o c h a l a s i n B ( C B ) ,
PMA and t e t r a c a i n e were o b t a i n e d
from Sigma C h e m i c a l s ( S t . L o u i s , MO, U.S.A). p r i l o c a i n e were donated P r o c a i n e was purchased
L i d o c a i n e and
by Fujisawa Chemicals Co.
Ltd.
(Osaka)
A l l of t h e l o c a l
from Hoei Yakko (Osaka).
a n e s t h e t i c s were used a s h y d r o c h l o r i d e s a l t s . P r e p a r a t i o n of PMN : P e r i p h e r a l
blood o b t a i n e d from h e a l t h y
v o l u n t e e r s was t r e a t e d w i t h h e p a r i n ( 2 0 U / m l of b l o o d ) and a pellet
c o n t a i n i n g PMN and
e r y t h r o c y t e s was s e p a r a t e d
whole b l o o d u s i n g a Lymphoprep ( N y e g a a r d C o . , d e n s i t y g r a d i e n t b y t h e method of Bb'yum ( 1 3 ) .
from
the
O s l o , Norway)
A PMN f r a c t i o n was
obtained by s e d i m e n t a t i o n of t h e p e l l e t i n a 1.5% (w/v) d e x t r a n (MW 1 7 7 , 0 0 0 ) - p h o s p h a t e b u f f e r e d s a l i n e ( P B S ) s o l u t i o n . Contaminating
erythrocytes i n
t h e PMN f r a c t i o n were removed
by
lysis w i t h a 0.75% ammonium c h l o r i d e s o l u t i o n c o n t a i n i n g 20 mM
Tris-HC1
b u f f e r ( f i n a l pH 7 . 4 ) f o r 10 m i n a t 37C w i t h g e n t l e
shaking.
T h e v i a b i l i t y of t h e PMN was t e s t e d b y t r y p a n b l u e
e x c l u s i o n and was found t o be g r e a t e r than 96%.
S A S AGA W A
6 10
Measurement of Migrating A c t i v i t y : M i g r a t i o n was measured u s i n g t h e modified Boyden chamber method ( 1 4 ) . PMN were s u s p e n d e d a t a c o n c e n t r a t i o n of 2 x 106 c e l l s / m l i n Hanks' balanced s a l t s o l u t i o n Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by Flinders University of South Australia on 01/12/15 For personal use only.
(HBSS) t h a t c o n t a i n e d 1% bovine s e r u m albumin and 0.3% NaHC03, and
were incubated i n a Boyden chamber (Bio-Rad f o r 45 m i n a t 37C u n d e r 5% C 0 2 .
Lab.,
Richmond, CA)
The migration d i s t a n c e (pm/45
c i n ) from t h e s u r f a c e of a M i l l i p o r e f i l t e r ( 3 . 0 p m pore s i z e ; M i l l i p o r e , Bedford, cells into
t o t h e l e a d i n g f r o n t of t h e
Mass, U.S.A)
i t was measured ( 1 5 ) .
T h e r e s u l t i n g v a l u e was u s e d t o
d e t e r m i n e c h e m o t a x i s t o FMLP
Local a n e s t h e t i c s i n
M).
v a r i o u s c o n c e n t r a t i o n s were added both
t o the upper
and
lower
compartments of t h e chamber immediately b e f o r e t h e a s s a y s . Lvsosomal Enzyme R e l e a s e from PMN : E x t r a c e l l u l a r r e l e a s e of t h e lysosomal enzyme, b e t a - g l u c u r o n i d a s e (BGL) was c a r r i e d o u t i n 200
p1 of t h e PMN s u s p e n s i o n (15).
Immediately a f t e r t h e a d d i t i o n of
M) a n d C B ( 5 p g / m l ) , t h e PMN s u s p e n s i o n a t a c o n c e n t r a t i o n of 2 x 106 c e l l s / m l i n HBSS was incubated f o r 15 m i n
FMLP
a t 37C.
Local a n e s t h e t i c s i n v a r i o u s c o n c e n t r a t i o n s were added t o
t h e c e l l suspension,
t o g e t h e r w i t h FMLP i n c o n j u n c t i o n w i t h CB.
The s u p e r n a t a n t and t h e PMN were s e p a r a t e d b y c e n t r i f u g a t i o n a t
1,700 x g f o r 2 m i n a t 4C.
BGL r e l e a s e d i n t o t h e s u p e r n a t a n t was
measured a c c o r d i n g t o a microassap m i x t u r e , which c o n s i s t e d
-
i n c u b a t e d f o r 60 m i n a t 37C.
The reaction
o f PMN s u p e r n a t a n t ,
methylumberlliferyl-beta-D-glucuronide, acetate buffer (pH 3.5)
(16).
method
1 mM 4-
0.1% T r i t o n X-100,
100 mM
a t o t a l v o l u m e o f 100 p 1 - w a s The r e a c t i o n was t e r m i n a t e d b y
a d d i t i o n of 2.4 m l of 50 mM g l y c i n e b u f f e r (pH 1 0 . 4 ) c o n t a i n i n g 5
m M EDTA.
F l u o r e s c e n c e was m e a s u r e b y
a
fluorometer
a t an
e x c i t a t i o n wavelength of 365 nm and an emission wavelength of 450 nm.
One u n i t o f t h e e n z y m e a c t i v i t y w a s e x p r e s s e d a s 4 -
m e t h y l u m b e l l i f e r o n e f o r m a t i o n i n pmol/min/mo of 2 x l o 6 PMN supernatant.
6 11
A N E S T H E T I C S I N HUMAN POLYMORPHONUCLEAR LEUKOCYTES
Measurement of SOA Production : SOA was assayed by t h e r e d u c t i o n of f e r r i c y t o c h r o m e c , u s i n g a s p e c t r o p h o t o m e t e r a t a wavelength of 550 nm (15).
The PMN s u s p e n s i o n a t a c o n c e n t r a t i o n of 1 x 106
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c e l l s / m l i n HBSS was p r e i n c u b a t e d f o r 10 m i n a t 37C i n a 1 m l
polystyrene cuvette.
After t h e a d d i t i o n of f e r r i c y t o c h r o m e c (66
pM; Sigma Chemicals, Type I V ) and v a r i o u s c o n c e n t r a t i o n s of l o c a l a n e s t h e t i c s , FMLP
M) i n c o n j u n c t i o n w i t h CB ( 5 p g / m l ) o r PMA
( 5 0 ng/ml) was added t o t h e c u v e t t e t o i n i t i a t e t h e r e a c t i o n .
The
c o n t i n u o u s r e d u c t i o n o f f e r r i c y t o c h r o m e c was measured.
The
maximum r e d u c t i o n r a t e was c a l c u l a t e d from t h e l i n e a r p o r t i o n of t h e d a t a which were recorded d u r i n g t h e i n i t i a l 2 m i n . The r a t e was e x p r e s s e d i n u n i t s ofAA550/min/ml of 1 x 106 PMN s u s p e n s i o n .
T e s t of C y t o t o x i c i t y o f L o c a l A n e s t h e t i c s t o PMN : PMN were incubated a t 37C f o r 15 m i n w i t h v a r i o u s c o n c e n t r a t i o n s of l o c a l anesthetics.
R e l e a s e of
t h e c y t o p l a s m i c enzyme l a c t a t e
dehydrogenase was determined by t h e method of Wacker e t a 1 ( 1 7 ) . T h e d e t e r g e n t T r i t o n X-100
(0.12,
s u p e r n a t a n t of t h e c e l l s u s p e n s i o n . a s p e r c e n t of t o t a l a c t i v i t y .
w/v) was a d d e d
to the
Enzyme a c t i v i t y was expressed
The t r y p a n b l u e e x c l u s i o n t e s t was
c a r r i e d out by a microscopic count of c e l l s (15).
T h i s test
involved c o u n t i n g c e l l s n o t s t a i n e d by 1% t r y p a n b l u e ,
and was
expressed a s percent of u n s t a i n e d c e l l s t o t o t a l c e l l n u m b e r . Noncytotoxic c o n c e n t r a t i o n s of l o c a l a n e s t h e t i c s ranged u p t o 10- 2
M f o r l i d o c a i n e , p r i l o c a i n e and p r o c a i n e , and 2.5 x
M for
t e t r a c a i n e ( d a t a not shown). S t a t i s t i c a l A n a l y s i s : T h e d a t a were r e p r e s e n t e d a s mean s t a n d a r d e r r o r (SE) f o r t h e combined e x p e r i m e n t s and d i f f e r e n c e s were analyzed f o r s i g n i f i c a n c e u s i n g t h e S t u d e n t ' s t - t e s t .
RESULTS The e f f e c t s of l o c a l a n e s t h e t i c s on chemotaxis of PMN t o 10- 8
M of FMLP a r e shown i n F i g u r e 1.
L i d o c a i n e , p r i l o c a i n e and
SASAGAWA
6 12
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120
I
"
-4.0
-3,6 -3.3 -3,O CONCENTRATION
(
-2.6
MOLAR
IN
-2.3
-2.0
LOGARITHM )
F i g u r e 1 : The e f f e c t s of l o c a l a n e s t h e t i c s on chemotaxis of PMN toward FMLP. PMN i n a Boyden chamber were i n c u b a t e d f o r 45 m i n a t 37C u n d e r 5%C02. The d i s t a n c e t h a t PMN migrated from t h e s u r f a c e i n t o a 3.0 um pore d i a m e t e r M i l l i p o r e f i l t e r was measured. Local a n e s t h e t i c s i n v a r i o u s c o n c e n t r a t i o n s were added both t o t h e u p p e r and lower comartments of t h e chamber i m m e d i a t e l y b e f o r e a s s a y . The r e s u l t i n g v a l u e was e v a l u a t e d a s percent t o t h e chemotaxis t o M of FMLP i n t h e a b s e n c e of l o c a l anesthetics. ( 0 )=lidocaine; ( 0 !=prilocaine; ( 0 >=procaine; ( )=tetracaine. Each p o i n t a d v e r t i c a l p-jlr represents t h e mean a n d SE f o r f o u r .F ~ ( 0 . 0 1l o c a l a n e s t h e t i c s v s c o n t r o l . e x p e r i m e n t s . -p
p r o c a i n e a t c o n c e n t r a t i o n s r a n g i n g from chemotaxis i n response t o FMLP (10- 8 M).
to
M inhibited
The i n h i b i t i o n s appeared
be c o n c e n t r a t i o n d e p e n d e n t and were s i g n i f i c a n t a t c o n c e n t r a t i o n s
of 5 x lo-'
M o r more.
T e t r a c a i n e a l s o showed a n i n h i b i t o r y
e f f e c t on c h e m o t a x i s , and a s i g n i f i c a n t e f f e c t was f o u n d a t c o n c e n t r a t i o n s of 5 x
and
M.
The e f f e c t s of t h e l o c a l a n e s t h e t i c s on BGL r e l e a s e from PMN exposed t o lom5 M of FMLP i n c o n j u n c t i o n w i t h 5 pg/ml of CB a r e shown i n F i g u r e 2.
Lidocaine a t c o n c e n t r a t i o n s of
to
M
613
ANESTHETICS I N HUMAN POLYMORPHONUCLEAR LEUKOCYTES
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150
100
-
be
Ln W
a W
_1 r
Wz
W -J
m 5c
I
I
-4.0
-3.3
1
-3,O
C O N C E N T R A T I O N ( MOLAR I N
I
-2.6
I
I
I
- 2 . 3 '-2.0
LOGARITHM
-2 I12
)
F i g u r e 2 : T h e e f f e c t s of l o c a l a n e s t h e t i c s on BGL r e l e a s e from PMN s t i m u l a t e d b y FMLP. L o c a l a n e s t h e t i c s i n v a i o u s c o n c e n t r a t i o n s were added t o PMN t o g e t h e r w i t h FMLP (10- M) i n c o n j u n c t i o n w i t h CB ( 5 pg/ml). A f t e r i n c u b a t i o n f o r 15 m i n a t 37C t h e a c t i v i t y of BGL r e l e a s e d from PMN was measured. T h e r e l e a s e d a c t i v i t y of BGL from s t i m u l a t e d PMN was e v a l u a t e d a s p e r c e n t t o t h a t i n t h e absence of t h e a g e n t . Each p o i n t and v e r t i c a l bar r e p r e s e n t s t h e mean and SE f o r f i v e experiments f o r l i d o c a i n e ( 0 ) and f o u r experiments,,for p r i l o c a i # g ( 0 ) , p r o c a i n e ( 0 ) , a n d tetracaine ( ). ~ ( 0 . 0 5 and ~ ( 0 . 0 1local anesthetics vs
< '
control.
inhibited
t h e r e l e a s e of
BGL from PMN.
Tetracaine
s i g n i f i c a n t i n h i b i t i o n a t c o n c e n t r a t i o n s of P r i l o c a i n e and p r o c a i n e were l e s s i n h i b i t o r y
caused
M o r more. compared
with
lidocaine o r tetracaine. F i g u r e 3 . shows t h e e f f e c t s of l o c a l a n e s t h e t i c s on SOA production of PMN induced by
M of FMLP i n c o n j u n c t i o n w i t h 5
SASAGAWA
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6 14
0
-11.3 -4.0
-3.6
-3.0
CONCENTRATION ( MOLAR I N
-2,3
-2.0
LOGARITHM
F i g u r e 3 : The e f f e c t s of l o c a l a n e s t h e t i c s on PMN SOA p r o d u c t i o n i n d u c e d b y FMLP i n c o n j u n c t i o n w i t h C B . L o c a l a n e s t h e t i c s i n v a r i o u s c o n c e n t r a t i o n s were a d d e d t o P N t o g e t h e r w i t h f e r r i c y t o c h r o m e c . A f t e r two m i n , FMLP ( 10- M ) i n c o n j u n c t i o n w i t h CB ( 5 pg/ml) was added t o PMN t o i n i t i a t e t h e r e a c t i o n . The maximum r e d u c t i o n r a t e of f e r r i c y t o c h r o m e c a t A550 was e v a l u a t e d a s p e r c e n t t o t h a t i n t h e absensce of t h e a g e n t . Each p o i n t and v e r t i c a l bar represents t h e mean and SE f o r f o u r experiments f o r p r i l o c a i n e ( 0 ) a n d t e $ f a c a i n e ( w ) & + f i v e f o r p r o c a i n e ( 0 ) and s i x f o r l i d o c a i n e ( 0 ). p