Bioscience Reports i, 89-99 (1981)
Printed in Great Britain
g9
M o l e c u l a r e v e n t s in l e u k o c y t e c h e m o t a x i s : t h e i r
p o s s i b l e r o l e s in p r o c e s s i n g t h e c h e m i c a l s i g n a l
Review
Elliott SCHIFFMANN
Laboratory of Developmental Biology and Anomalies,
National Institute of Dental Research, National Institutes
of Health, Bethesda, Maryland 20205, U.S.A.
(Received 19 February 1981)
C h e m o t a x i s , t h e d i r e c t e d migration of cells along a gradient of
a t t r a c t a n t , has been studied in a variety of ceils. Bacteria respond to
simple nutritive molecules such as sugars and amino acids (1~2); the
c e l l u l a r s l i m e molds to c y c l i c n u c l e o t i d e s
(3); leukocytes
to
w e l l - d e f i n e d c o m p o u n d s such as d e r i v a t i v e s of c o m p l e m e n t ( 4 ) ,
Iormylated peptides (5,6)~ and metabolites of a r a c h i d o n i c aci d ( 7 ) ;
tumor cells to c o m p l e m e n t - r e l a t e d substances (8); and neuronal ceils
to nerve growth f a c t o r (9).
Quite r e c e n t l y , a n u m b e r of c o n n e c t i v e - t i s s u e - t y p e ceils have been shown to migrate toward a t t r a c t a n t s
of varying degrees of c h a r a c t e r i z a t i o n . These include fibroblasts (10),
smooth-muscle cells ( l l ) , and endothelial ceils (12).
In those ceils in which chemotaxis has been extensively studied, the
process has clearly been shown to be initiated by interaction between
an a t t r a c t a n t and a specific receptor on the membrane - the putative
r e c o g n i t i o n of the chemical signal (13-15).
The biochemical events
which intervene b e t w e e n signal r e c o g n i t i o n and a c t i v a t i o n of t h e
motile elements of the cell are presently the locus of intense interest,
for it is becoming evident t h a t such p r o c e s s e s may h a v e c o m m o n
f eatu r es in a variety of r e c e p t o r - m e d i a t e d responses (16).
While the
g r e a t e s t progress in this area has o c c u r r e d in s t u d i e s of b a c t e r i a l
chemotaxis, we shall consider here such aspects of leukocyte chemotaxis: these cells, whose motile response is crucially important in the
defense of the host, apparently utilize certain biochemical reactions of
the membrane in amplifying the signal.
The availability of both well-defined form yl at ed peptides (5,6) and
the completely c h a r a c t e r i z e d complement d e r i v a t i v e CSa ( 1 7 ) , e a c h
a c t i v a t i n g d i f f e r e n t receptors (13,18), has facilitated studies on the
b i o c h e m i s t r y of l e u k o t a x i s .
In T a b l e 1 a r e l i s t e d a v a r i e t y of
r e c e p t o r - m e d i a t e d b e h a v i o r a l and m o l e c u l a r c o n s e q u e n c e s of the
exposure of leukocytes to a t t r a c t a n t s .
The phenomena of adherence,
s p r e a d i n g , o r i e n t a t i o n , and l o c o m o t i o n a r e a l l n e c e s s a r y f o r the
integrated c h e m o t a c t i c r e s p o n s e .
Som e of t h e m o l e c u l a r e v e n t s
( c y c l i c AMP formation, protein carboxymethyl esterification~ changes
in m e m b r a n e p o t e n t i a l ) o c c u r r a p i d l y enough to be p a r t of t h e
t r a n s d u c t i o n sequence in signal amplification, but may also modulate
the c h e m o t a c t i c response.
Others (e.g. hexose monophosphate shunt)
provide energy for motility.
I shall discuss these and other r e c e p t o r mediated functions in an a t t e m p t to define t h e i r r o l e s in e f f e c t i n g
leukocyte chemotaxis.
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The Biochemical Society
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SCHIFFMANN
Table i.
Functions stimulated in neutrophils
exposed to chemoattractants
Behavioral
Adherence
Spreading
Orientation
Locomotion (random and directed)
Phagocytosls and killiag
Release of lysosomal enzymes
Molecular
Active oxygen production (H202, 02- , 02+)
Cyclic nucleotide changes
Changes in methylated phospholipids and protein methyl e s t e r s
Hexose monophosphate shunt
A variety of membrane-associated reactions:
hyperpolarization, increased cationic fluxes, redistribution
of calcium, and decreased cell surface charge
Internalization of Agonist-Receptor Complex
In earlier studies on the initial interaction between formyl peptide
a t t r a c t a n t s and n e u t r o p h i l s (19)7 it was suggested that a peptidase
associated with the m e m b r a n e r e c e p t o r h y d r o l y z e d bound peptide~
s e r v i n g to f r e e t h e binding site a f t e r the chemical signal has been
initiated.
Conceivably~ such a p r o c e s s m i g h t e n a b l e t h e c e l l to
d e t e c t successively new molecules of a t t r a c t a n t as it migrated along
the gradient. However~ r ecent studies have clearly shown that a more
complex picture exists.
T h r e e groups h a v e d e m o n s t r a t e d t h a t
r e c e p t o r - m e d i a t e d endocytosis of labeled ligand occurs in a t i m e- and
t e m p e r a t u r e - d e p e n d e n t p r o c e s s (15,20,21).
In intervals of time as
short as 2 min a f t e r exposure of cells to l a b e l e d p e p t i d e a t 2 # ~
s o m e 50% of s p e c i f i c a l l y bound a t t r a c t a n t could not be dissociated
f r o m t h e cel l .
F u r t h e r m o r e , with l o n g e r e x p o s u r e , a p p r e c i a b l e
h y d r o l y s i s of the peptide occurred, most likely within the cell since
considerable r a d i o a c t i v i t y was f ou nd in t h e l y o s o m a l f r a c t i o n of
disrupted cells (21).
N i e d e l e t a 2 . (22) h a v e p r o v i d e d visual
evidence of internalization of fluorescent peptide% a process occurring
within 5 min.
Cells exposed to unlabeled peptide a t t r a c t a n t show a
d e c r e a s e in binding s i t e s when t e s t e d s u b s e q u e n t l y w i t h l a b e l e d
attractant (21).
R e c o v e r y of binding occurs when the stimulus is
removed. In the case of t h e C5a r e c e p t o r , r e c o v e r y is p a r t i a l l y
i n h i b i t e d by inhibition of protein synthesis.
The neutrophil r e c e p t o r
exhibits the phenomenon of deactivation, or ' d o w n - r e g u l a t i o n ' , w hi ch
has been observed for other receptors.
Although the relatively rapid time periods during which i n t e r n a l i z a t i o n of t h e p u t a t i v e ligand-receptor complexes occurs may merit
their consideration as early events in the transduction process, s o m e
r e c e n t r e s u l t s a r g u e a g a i n s t this conclusion. One set of evidence
concerns the relationship betwen 'down-regulation' and 'deactivation' of
PROCESSING THE SIGNAL IN LEUKOCYTE CHEMOTAXIS
91
n e u t r o p h i l chemotaxis.
Donabedian and Gallin (23) have observed a
non-preferential chemotactic deactivation induced by the complementderived attractant.
That is, prior exposure of ceils to this attractant
reduced the c h e m o t a c t i c response to i t s e l f and to a I o r m y l a t e d
peptide.
Yet the number ol binding sites for radiolabeled formylated
peptide (fMet-Leu-ESH]Phe) was actually increased, or 'up-regulated'.
On the o t h e r hand, c e l l s exposed to fMet-Leu-Phe were transiently
deactivated to this peptide, but not to the C5 attractant, and showed
a loss of binding sites for the peptide.
In another study, Niedel e t
a l . (24) found that inhibitors of chymotryptic a c t i v i t y reduced uptake
(including internalization) of labeled peptide at concentrations of two
orders of magnitude greater than those required to inhibit neutrophil
chemotaxis. Also, Warabi at a l . (in preparation) found that dansylcadaverine, used by o t h e r s to i n h i b i t i n t e r n a l i z a t i o n of e p i d e r m a l
g r o w t h f a c t o r and c~2-macroglobulin (25,26), almost t o t a l l y reduced
chemotaxis at concentrations which had no effect upon internalization.
A t higher levels~ the reagent did inhibit internalization.
At present, the relationship between a r e c e p t o r - m e d i a t e d loss in
chemotacts
responsiveness and changes in binding of an attrac:tant to
its receptor is not clear. Indeed, these results suggest that receptorm e d i a t e d i n t e r n a l i z a t i o n of the ligand and the chemotactic response
are not t i g h t l y coupled and, therefore, that the subsequent endocytic
e v e n t s are not on the direct sequence of signal transduction.
They
may, however, have a role in a d a p t a t i o n of the c e l l to a given
stimulus.
Changes in Membrane Potential
C h e m o a t t r a c t a n t s induce changes in membrane potential rapidly
enough to justify their consideration as e a r l y events in the t r a n s d u c t i o n of the signal (27,28).
What appears to be a depolarization
occurs first, followed by a hyperpolarization of the membrane.
The
latter phenomenon, detected within the first minute after chemotactic
stimulation, is definitely associated with an increase in permeability to
K + and is expressed maximally in the presence ~of extracelluJar Ca2+
(29).
It is of interest that neutrophiJs from p a t i e n t s w i t h c h r o n i c
g r a n u l o m a t o u s d i s e a s e ( C G D ) s h o w e d a m a r k e d r e d u c t i o n in the
stimulated changes in membrane potential com pared to n o r m a l c e l l s
(28).
The p a t i e n t s ' ceils are deficient in active oxygen production
and in the ability to kill phagocytized bacteria.
However, these ceils
possess at l e a s t 60% of t h e c h e m o t a c t i c responsiveness of normal
cells. Addition of active oxygen-generating systems to CGD cells did
n o t r e s t o r e stimulated membrane potential changes or chemotaxis to
normal , nor, on the other hand, did addition of a c t i v e o x y g e n s c a v e n g e r s or s u p e r o x i d e d i s m u t a s e c h a n g e t he m em brane response in
n o r m a l c e i l s e x p o s e d to a t t r a c t a n t .
These results suggest that
alterations in membrane potential, although they are rapid events, may
be more related to oxygen metabolism than to direct processing of the
c h e m o t a c t i c signal; they may well modulate it.
Ionic Events
Leukocyte chemotaxis, as measured in the modified Boyden c:hamber
(30), is optimal in the presence of external Na +, K+, Ca 2+, and Mg2+.
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5CHIFFMANN
Chemoattractants
h a v e b e e n f o u n d to s t i m u l a t e a m e m b r a n e - b o u n d
Na +, K + - A T P a s e with a t t e n d a n t rapid c h a n g e s in t h e f l u x e s of b o t h
m o n o v a l e n t c a t i o n s and Ca 2+ ( 3 1 ) .
The A T P a s e a p p e a r s to function
as a m o n o v a l e n t cation pump.
R e c e n t studies h a v e provided e v i d e n c e
t h a t a t l e a s t t w o t y p e s of C a e+ m o v e m e n t are s t i m u l a t e d in the
p r e s e n c e of a t t r a c t a n t s : an i n c r e a s e in cell p e r m e a b i l i t y to C a 2+ and a
r e l e a s e of p r e s u m a b l y m e m b r a n e - b o u n d cation to the c y t o p l a s m (31).
The f o r m e r is s u p p o r t e d by d e m o n s t r a t i o n s of both i n c r e a s e d u p t a k e
and steady-state
l e v e l s of 45Ca2+ in s t i m u l a t e d cells; the l a t t e r by
o b s e r v a t i o n s of the d e c r e a s e in f l u o r e s c e n c e
in c h l o r t e t r a c y c l i n e t r e a t e d ceils ( 3 2 ) .
Both of t h e s e changes a r e d e t e c t a b l e within one
minute after stimulation.
It is of i n t e r e s t t h a t the s t i m u l a t e d i n c r e a s e
in p e r m e a b i l i t y
to C a 2+ is i n h i b i t e d by a g e n t s which depress the
c o n v e r s i o n of a r a c h i d o n i c a c i d to o x y g e n a t e d d e r i v a t i v e s v i a t h e
lipoxygenase pathway.
H o w e v e r , the s t i m u l a t e d r e d i s t r i b u t i o n of Ca 2+
is not. As will be c o n s i d e r e d l a t e r , such a r a c h i d o n a t e m e t a b o l i t e s m a y
play s i g n i f i c a n t roles in the c h e m o t a c t i c response. In an i n t e r p r e t a t i o n
of t h e s e results, B e c k e r a n d S t o s s e l l ( 3 3 ) h a v e s u g g e s t e d t h a t a
p r i m a r y e v e n t in c h e m o t a c t i c s t i m u l a t i o n is the r e l e a s e of m e m b r a n e bound Ca 2+ which a c t i v a t e s phospholipase A2, l i b e r a t i n g a r a c h i d o n a t e .
This, in turn, is c o n v e r t e d to m e t a b o l i t e s which a c t to i n c r e a s e cell
p e r m e a b i l i t y to C a 2+, w h i c h c a t i o n , in a m a n n e r n o t y e t c l e a r ,
c o n t r i b u t e s to the i n t e g r a t e d c h e m o t a c t i c r e s p o n s e as well as to o t h e r
neutrophil functions such as s e c r e t i o n of l y s o s o m a l e n z y m e s (3t~).
Cyclic Nucleotide Changes
A n u m b e r of studies (35,36) on the role of c y c l i c n u c l e o t i d e s in
c h e m o t a x i s have i n d i c a t e d t h a t a g e n t s w h i c h s t i m u l a t e
cyclic AMP
f o r m a t i o n inhibit c h e m o t a x i s , while a g e n t s which s t i m u l a t e c y c l i c GMP
formation enhance chemotaxis.
In t h e s e s t u d i e s , c h a n g e s in c y c l i c
n u c l e o t i d e s s t i m u l a t e d by c h e m o a t t r a c t a n t s t h e m s e l v e s w e r e not well
c o r r e l a t e d with the c h e m o t a c t i c responses e l i c i t e d by t h e a t t r a c t a n t
(37).
The d e t e r m i n a t i o n s of c y c l i c n u c l e o t i d e c h a n g e s w e r e p e r f o r m e d
g e n e r a l l y at i n t e r v a l s g r e a t e r than one m i n u t e a f t e r e x p o s u r e of cells
to attractant.
In f u r t h e r s t u d i e s t h a t h a v e f o c u s e d upon s h o r t e r
i n t e r v a l s , a n u m b e r of g r o u p s ( 3 8 - t ~ 1 ) h a v e o b s e r v e d t h a t c h e m o attractants
i n d u c e d a rapid, t r a n s i e n t t w o - to t h r e e - f o l d i n c r e a s e in
c y c l i c AMP, beginning a t 5 to 10 s a f t e r s t i m u l a t i o n and r e t u r n i n g to
basal levels within 5 min.
C h a n g e s in cyclic GMP, on the o t h e r hand,
could n o t be d e t e c t e d
within these intervals.
Other stimulated
l e u k o c y t e f u n c t i o n s s u c h as s u p e r o x i d e p r o d u c t i o n and r e l e a s e of
l y s o s o m a ! e n z y m e s o c c u r r e d s u b s e q u e n t l y and could be uncoupled f r o m
the stimulated
i n c r e a s e in c y c l i c AMP.
T h a t is, addition of a g e n t s
which raised c y c l i c AMP levels ( m e t h y l x a n t h i n e s , p r o s t a g l a n d i n E
[ P G E ] ) a b r o g a t e d the s t i m u l a t e d l e u k o c y t e functions and, in the c a s e
of PGE, i n d e p e n d e n t l y r a i s e d t h e l e v e l of c y c l i c AMP a b o v e t h a t
produced by a t t r a c t a n t alone.
T h e s e c o n f l i c t i n g results h a v e not y e t
been resolved.
As S i m c h o w i t z e t a l . (39) pointed out, it is possible
t h a t the c h e m o t a x i s - r e c e p t o r - m e d i a t e d c h a n g e s in c y c l i c AMP m a y be
c o m p a r t m e n t a l i z e d in the cell c o m p a r e d to t h e p r e s u m a b l y ' g l o b a l '
c h a n g e s induced by drugs.
The f o r m e r e v e n t m a y be required for the
cellular response, while the l a t t e r m a y be m o d u l a t o r y .
PROCESSING THE SIGNAL IN LEUKOCYTE CHEMOTAXIS
93
Th e r a p i d i t y of t h e s t i m ul at ed increase in cyclic AMP has been
observed in another c h e m o a t t r a c t a n t - i n d u c e d change.
Wedner e t a l .
( 4 2 ) h av e f ound t h a t i n c r e a s e d p h o s p h o r y l a t i o n of a 90-kilodalton
protein occurred within 5 s, the increase decaying to c o n t r o l l e v e l s
w i t h i n 10 rain.
These results are consistent with a c h e m o t a c t i c a l l y
stimulated cyclic AMP-dependent protein kinase in the early stages of
signal transduction.
It is of interest that this stimulated phosphorylation could be inhibited by agents which block arachidonate metabolism
when the c h e m o a t t r a c t a n t s were peptides (C5a and f M e t - L e u - P h e ) , but
n o t the oxidized arachidonate d e r i v a t i v e 5 - h y d r o x y - e i c o s a t e t r a e n o i c
acid (5-HETE). Present evidence indicates that stimulated formation
of 5-HETE (43) occurs later than changes in cyclic AMP, suggesting
that the l a t t e r events may not be essential for protein phosphorylation.
Although the c h a r a c t e r i s t i c s of c y c l i c AMP c h a n g e s ( r e c e p t o r mediated,
r a p i d , t r a n s i e n t , p r o x i m a l to c e l l u l a r r e s p o n s e s ) a r e
c o n s i s t e n t with w ha t may be r e q u i r e d in e a r l y e v e n t s of signal
t r a n s d u c t i o n , p r e s e n t e v i d e n c e still f a v o r s t h e i r c o n s i d e r a t i o n as
regulatory processes rather than as essential ones in chemotaxis.
Turnover of P r o t e i n Methyl E s t e r s
In b a c t e r i a l c h e m o t a x i s , t h e i n v o l v e m e n t of p r o t e i n m e t h y l
e s t e r i f i c a t i o n and its hydrolysis has been clearly shown (44,g5).
The
cell registers its adaptation to a given level of stimulus by a change
in t h e a m o u n t of specific, m e t hylat ed membrane proteins.
In some
manner, this process plays a role in transducing the chemical signal to
the cell's motility elements. In studies with rabbit neutrophils, O'Dea
e t a l . (46) reported that f or m yl a ted peptide a t t r a c t a n t s specifically
stimulated the incorporation of labeled methyl group from methionine
into protein methyl e s t e r s in a rapid (30 s to 1 m i n ) , t r a n s i e n t
manner.
In s u b s e q u e n t s t u d i e s , V e n k a t a s u b r a m a n i a n e t a l . ( g 7 )
reported a stimulated hydrolysis of labeled methyl esters from proteins
in t h e s e c e l l s .
These results suggested that chemoattractants
stimulate turnover of protein methyl esters, a process which may be
l i n k e d t o signal t r a n s d u c t i o n as in b a c t e r i a .
Conceivably, the
reversible methylation could lead to critical conformational changes in
a membrane protein and in some manner a f f e c t motility.
However,
these events have not been dem onst rat ed either in human neutrophils
or in monocytes. In addition, the great lability of these methyl esters
from animal cells has so far precluded a t t e m p t s to i d e n t i f y s p e c i f i c
proteins that may participate in chemotaxis through methylation. The
role of protein methyl e s t e r i f i c a t i o n in leukocyte chemotaxis is not yet
clear.
M e m b r a n e Lipid R e a c t i o n s in C h e m o t a x i s
Phospholipid turnover
Pike e t a l . (48) demonstrated that agents which raise intracellular
levels of S-adenosylhomocysteine, a potent m e t h y l t r a n s f e r a s e inhibitor,
m a r k e d l y depressed chemotaxis, as welt as both phospholipid methylation and protein carboxylmethylation in monocytes.
Similar findings
h a v e b een o b s e r v e d f or t h e n e u t r o p h i l (49).
Direct evidence for
94
SCHIFFMANN
changes in lipid methylation in c h e m o t a c t i c a l l y stimulated neutrophils
was provided by Hirata e t a l . (43). They demonstrated a stimulated
d e c r e a s e of t he incorporation of the [3H]methyl group from labeled
methionine into phosphoJipids, but did not find significant c h a n g e s in
t h e l e v e l s of p h o s p h o l i p i d s ar i s i ng f r o m the CDP-choline pathway.
This suggested that the transmethylation pathway in lipid metabolism,
t h o u g h q u a n t i t a t i v e l y subordinate to the synthetic one, could play a
role in n e u t r o p h i l c h e m o t a x i s .
In t h e s e s t u d i e s , t h e a t t r a c t a n t s t i m u l a t e d d e m e t h y l a t i o n r eact i on was correl at ed well with a rapid
release of arachidonate, presumably via phospholipase A2 a c t i n g upon
phosphatidylcholine.
In a m o r e recent study (5areis e t a l . , subm i t t e d ) , r e c e p t o r - m e d i a t e d stimulation of arachidonic acid r e l e a s e in
n e u t r o p h i l s was found to be inhibited by m e t h y l t r a n s f e r a s e inhibitors
while Ca2+-ionophore-induced release was not.
The stimulated i n f l u x
of Ca 2+ by a t t r a c t a n t s was also reduced by inhibitors of methylation.
These results indicate a role for phospholipase A2 (a C a 2 + - r e q u i r i n g
en zy me) in leukocyte chemotaxis and suggest that this reaction may
be part of a t u r n o v e r process for m e t h y l a t e d p h o s p h o l i p i d s t h a t is
necessary for the integrated c h e m o t a c t i c response. In accordance with
this suggestion is the finding t h a t bot h c h e m o t a x i s and s t i m u l a t e d
r e l e a s e of a r a c h i d o n i c aci d in n e u t r o p h i l s w e r e i n h i b i t e d in the
presence of mepacrine, an inhibitor of the lipase (43).
These events
a p p e a r to be c o m m o n in other r e c e p t o r - m e d i a t e d processes such as
IgE-stimulated release of histamine in mast cells and basophils (16).
However, in platelets the activation of these cells appears to involve
stimulation of a phosphatidylinositol-specific p h o s p h o l i p a s e - C a c t i v i t y
prior to the release of arachidonate from diacylglycerol (50).
In macrophages, Snyderman e t a l . (51) have found t h a t c h e m o a t t r a c t a n t s inhibit methylation of phospholipids but, unlike their e f f e c t
in neutrophils, do not stimulate their d e g r a d a t i o n .
Although t h e r e
appear to be differences in the nature of lipid transformations between
these c h e m o t a c t i c a l l y responsive leukocytes, the studies on both ceils
are consistent with the idea that r e c e p t o r - m e d i a t e d localized changes
in membrane lipid compositions may cause alterations in fluidity of the
m e m b r a n e , which in t u r n are linked in some manner to the motile
response (43,52).
Regulation of phospholipase A 2
T h e l e u k o c y t e apparently can regulate its c h e m o t a c t i c responsiveness by af f e c t i ng phospholipase A2 activity.
Evidence for this is in
the demonstration that glucocorticoids, classic anti-inflammatory
agents, induce synthesis of a phospholipase inhibitor in a n u m b e r of
tissues, including leukocytes (53,5#). Hirata e t a l . ( 5 # ) showed that
t r e a t m e n t of these cells with steroids such as dexamethasone resulted
in s y n t h e s i s de n o v o of a 40-kilodalton protein that inhibited both
leukocyte phospholipase A2 and chemotaxis.
A similar protein that
inhibited prostaglandin generation has been reported to be produced in
cultured macrophages (55).
Since such a protein is present at basal
levels in leukocytes not t r e a t e d with glucocorticoids, it is possible that
c h e m o t a c t i c activation of phospholipase may involve the inactivation of
endogenous inhibitor.
Some indication for such an o c c u r r e n c e may be
in the findings that Ca 2+ partially reverses the inhibition of phospho-
PROCESSING THE SIGNAL IN LEUKOCYTE CHEMOTAXIS
95
lipase A2 by the steroid-induced inhibitor (56) and that c h e m o a t t r a c t a n t s s t i m u l a t e t h e p h o s p h o r y l a t i o n of this i n h i b i t o r in a c y c l i c
A M P - d e p e n d e n t r e a c t i o n (F. H i r a t a , submitted).
It remains to be
demonstrated whether the phosphorylated inhibitor is inactive.
If so,
this f in d in g could i n d i c a t e a r ole for the r e c e p t o r - m e d i a t e d early
stimulation of cyclic AMP formation as well as an additional function
for the stimulated Ca 2+ redistribution reaction (32).
Metabolism of arachidonic acid
Th e c h e m o t a c t i c a l l y s t i m u l a t e d r e l e a s e of a r a c h i d o n i c acid in
neutrophils has been shown to result in f o r m a t i o n of p r o s t a g l a n d i n s
(57,58) and a variety of oxidized derivatives of the f a t t y acid (59), a
principal one being 5-HETE, itself an a t t r a c t a n t .
I n h i b i t o r s of t hi s
l i p o x y g e n a s e p a t h w a y f or a r a c h i d o n a t e m e t a b o l i s m were found to
reduce r e c e p t o r - m e d i a t e d functions of the leukocyte such as c h e m o t a x i s and l y s o s o m a l e n z y m e release (60), whereas inhibitors of the
cyclooxygenase pathway had little e f f e c t . These results suggested that
an o x i d i z e d d e r i v a t i v e of a r a c h i d o n a t e formed via the lipoxygenase
pathway may be required for leukocyte functions.
T h e p o s s i bl e r ol e of m e t a b o l i t e s of a r a c h i d o n a t e in leukocyte
chemotaxis has been extensively studied by Goetzl and c o l l a b o r a t o r s
(61-63).
A variety of the HETE compounds, produced in neutrophils
by c h e m o t a c t i c s t i m u l a t i o n , a r e t h e m s e l v e s
leukoattractants;
5 , 1 2 - d i H E T E is t h e m os t p o t e n t , a c t i v e in t h e n a n o m o l a r range.
Analysis of s u b f r a c t i o n s of d i s r u p t e d n e u t r o p h i l s in which l a b e l e d
HETEs had been g e n e r a t e d i n d i c a t e d a preferential localization oi
t h e s e c o m p o u n d s in cel l m e m b r a n e s .
T r e a t m e n t of c e i l s w i t h
i n h i b i t o r s of t h e l i p o x y g e n a s e pathway for arachidonate metabolism
resulted in depletion of 5- and l l-HETE from cell membranes and also
d e p r e s s e d c h e m o t a x i s to other a t t r a c t a n t s (CSFr and fMet peptide).
This inhibition of chemotaxis could be overcome either by the addition
of 5- and I I-HETE to cells in the presence of inhibitor or by washing
out the inhibitor.
Restoration of c h e m o t a x i s was a c c o m p a n i e d by
r e s t o r a t i o n of i n t r a c e l l u l a r levels of HETEs. Of interest here is a
very r ecen t report that c h e m o a t t r a c t a n t s stimulate a rapid, transient
i n c o r p o r a t i o n of label from [SH]arachidonate into a p r o t e i n fraction
from neutrophils (D. Goldman & E.3. Goetzl, personal communication).
T h e s e r e s u l t s s u g g e s t t h a t l i p o x y g e n a s e - g e n e r a t e d m e t a b o l i t e s of
arachidonic acid may have two roles in l e u k o c y t e c h e m o t a x i s : t h e y
may be endogenous a t t r a c t a n t s which upon their release from the cell
would amplify the c h e m o t a c t i c response initiated by external a t t r a c t a n t s , and t h e y m a y C o n t r i b u t e to processing the signal in the cell
membrane.
This l a t t e r role is supported by the studies of Naccache
e e a 2 . ( 6 t t ) , who found t h a t l i p o x y g e n a s e i n h i b i t o r s d e p r e s s e d
a t t r a c t a n t - s t i m u l a t e d Ca 2+ influx.
A c t i v a t i o n of C y t o s k e l e t a l
Elements
C h e m o a t t r a c t a n t s stimulate the assembly of microtubules and actin
filaments in leukocytes (65), but there is no evidence on the nature
of the molecular events which link activation of the cytoskeleton to
any of the possible transduction reactions discussed.
A recent study
96
SCHIFFMANN
Table 2.
Possible sequence of molecular events in
transducing the leukotactic signal
A. Interaction of chemoattractant
with membrane receptor generates
a signal, resulting in subsequent steps.
of Ca 2+ occurs: membrane-bound Ca 2+ is released
into the cytosol.
Speculation: Ca 2+ may be bound to protein
carboxyl groups and released by stimulated methylation of the
carboxyls.
B. Redistribution
C. M e t h y l a t i o n
of phospholipids
and activation
of phospholipase
A2, leading to release of arachidonic acid. May be enhanced by
rapid increase in cyclic AMP, activating a kinase to inactivate
endogenous inhibitor of the enzyme.
D. Conversion of arachidonic acid to HETEs.
E. Ca 2+ influx is stimulated: may be required for microtubule
assembly and contractile events of actin and myosin.
Ca 2+
influx in part may depend upon phospholipid methylation.
i n d i c a t e s t h a t c h e m o t a c t i c s t i m u l a t i o n of neutrophils c a u s e s e n h a n c e d
t y r o s y l a t i o n of tubulin, a p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n of this protein
(66).
It is not known, h o w e v e r , w h e t h e r such an e v e n t is required for
a s s e m b l y of tubulin into m i c r o t u b u l e s or for e f f i c i e n t c h e m o t a x i s .
The
studies
of S t o s s e l a n d his c o l l a b o r a t o r s
have yielded important
i n f o r m a t i o n on the n a t u r e of l e u k o c y t e c o n t r a c t i l e p r o c e s s e s involving
actin and myosin in vitro
(67,68).
Y e t , the d e m o n s t r a t i o n t h a t
e v e n t s such as r e v e r s i b l e cross-linking of a c t i n o c c u r i n v i v o a w a i t s
further investigation.
A Possible
Leukocytes
Sequence
in P r o c e s s i n g the C h e m o t a c t i c
Signal in
In Table 2 are given steps in a possible sequence of biochemical
events in processing the chemotactic signal, A number of modulatory
mechanisms
may also operate.
At A, the i n t e r a c t i o n of a t t r a c t a n t
with r e c e p t o r can be a f f e c t e d by the s u b s e q u e n t d o w n - r e g u l a t i o n
of
the receptor.
At r
i n c r e a s e s in c y c l i c AMP h a v e been a s s o c i a t e d
m o r e with i n h i b i t i o n
of c h e m o t a x i s than with s t i m u l a t i o n , a d i s c r e p a n c y s t i l l to be resolved.
At D, a r a c h i d o n a t e m a y be c o n v e r t e d to
5,12-diHETE, a p o t e n t l e u k o a t t r a c t a n t t h a t m a y a m p l i f y t h e c h e m o t a c t i c response evoked by the p r i m a r y stimulus.
In B, the influx of
C a 2+ m a y be required for c e r t a i n c h a n g e s in m e m b r a n e p o t e n t i a l , the
a b s e n c e of which is a s s o c i a t e d with a b n o r m a l c h e m o t a x i s .
Conclusions
Chemotaxis
is an e x a m p l e of c e l l b e h a v i o r in which a t h e r m o dynamically favorable occurrence, agonist-receptor interaction, initiates
profound c h a n g e s in the cell, mobilizing and utilizing l a r g e a m o u n t s of
energy.
The s t i m u l a t e d t u r n o v e r of m e t h y l a t e d
phospholipids and
s u b s e q u e n t t r a n s f o r m a t i o n s of a r a c h i d o n i c acid a p p e a r to be crucial
PROCESSING THE SIGNAL IN LEUKOCYTE CHEMOTAXI5
97
events in the amplification of this r e c e p t o r - m e d i a t e d process, leading
to the motile response.
Methylation, t h e r e f o r e , may play a rol e as
s e c o n d m e s s e n g e r in chemotaxis as it appears to do in a variety of
receptor-mediated processes (16).
A c o m m o n f e a t u r e of t h e s e
r e s p o n s e s is t h e i r r e q u i r e m e n t for Ca 2+, and this discussion has
indicated some reactions involving methylation which appear to prime
the cell for its unique Ca2+-triggered functions.
An understanding of
the intervening events in such r e c e p t o r - m e d i a t e d r e s p o n s e s in t h e
leukocyte might well apply to motile behavior observed in a wtriety of
non-phagocytic cells (10,12).
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