(1996) Immunopharmacology
ELSEVIER
Immunopharmacology32 19-23
Neutrophil adhesion on surfaces preadsorbed with high molecular
weight kininogen under well-defined flow conditions
L in-Yue L. Y ung a, F lorencia L im a, M ohammad M.H. Khan b, Satya P. Kunapuli b,
Leonard R ick b, Robert W. C olman b, Stuart L. C ooper a
~' Department o f Chemical Engineering, Uni~:ersity o f Delaware, Newark, DE 19716, USA
Sol Sherry Thrombosis Research Center, Temple UnicersiO, School o f Medicine, Philadelphia, PA 19140, USA
A bstract
T he a dhesion o f n eutrophils a nd o ther l eukocytes to b iomaterial s urfaces is an i mportant p henomenon in the h ost
r esponse to b iomaterials b ecause t he n umber o f a dherent l eukocytes is o ften r elated to the i nflammatory r esponse a fter
i mplantation. A fter a dhering t o b iomaterial s urfaces, o ther l eukocyte r eactions, such as p hagocytosis, r espiratory burst, a nd
p rotease r elease, m ay o ccur a nd r esult in t he d eterioration o f t he i mplanted b iomaterial a nd i njury to p eripheral tissue. T his
s tudy o f n eutrophil a dhesion q uantitatively c haracterizes n eutrophil a dhesion under w ell-defined l aminar f low c onditions
u sing a r adial f low c hamber. I n t his r heologically w ell-defined s ystem, t he f luid s hear r ate o n t he s urface v aries c ontinuously
w ith r adial p osition. T his a llows t he s tudy o f s hear-dependent b ehavior o f n eutrophil a dhesion. E xploiting t he v ariable s hear
r ate in t he r adial f low c hamber, t he k inetics o f n eutrophil a dhesion was o btained u sing a utomated v ideo m icroscopy and
i mage a nalysis to r ecursively a cquire c ell c ounts f rom m ultiple f ields in d ifferent r adial p ositions, a nd t o q uantify t he s urface
d ensity o f n eutrophil as a f unction o f t ime, N eutrophil a dhesion w as s tudied o n g lass p readsorbed w ith f ibrinogen a nd
h igh-molecular-weight k ininogen ( HK). A t a s hear r ate o f 20 s - t , t he n umber o f a dherent c ells on t he p readsorbed
f ibrinogen s urface w as s imilar to t hat on b are g lass, a nd t he n umber o f a dherent c ells o n t he H K s urface w as l ess than 10%
o f t hat on the b are g lass. W e c onclude t hat s urfaces p readsorbed w ith H K a re a nti-adhesive to n eutrophils.
Keyword.s: Neutrophil adhesion; High molecular weight kininogen; Radial flow system
1 . I ntroduction
B iomaterials h ave b een e xtensively u sed i n c linic al a pplications i n r ecent y ears i n b oth t emporary
d evices a nd p ermanent i mplants. T wo b asic c riteria
a re r equired f or a s uccessful b iomaterial: ( 1) t he
m aterial h as t o b e b iocompatible t o t he e xtent t hat
t he a dverse h ost r esponses t hat a rise d ue t o t he
p resence o f t he i mplant a re m inimized; ( 2) t he m ater ial s hould p ossess g ood p hysical a nd m echanical
p roperties a nd b e a ble t o r esist d eterioration d uring
e xposure t o t he b iological e nvironment. L eukocyte
a dhesion t o b iomaterial s urfaces i s a n i mportant f act or i n t he h ost r esponse t o b iomaterials b ecause t he
n umber o f a dherent l eukocytes i s o ften r elated t o
t heir i nflammatory r esponse a fter i mplantation [ 1,2].
L eukocyte a dhesion t o p olymer s urfaces i s a lso
k nown t o o ccur d uring m any t ypes o f p rocesses
i nvolving b l o o d - m a t e r i a l i nteractions s uch a s
h emodialysis [ 3-5], c ardiopulmonary b ypass [6], a nd
a rtificial h eart i mplantation [7]. I n a ddition, l ittle
q uantitative w ork h as b een d one o n t he i nteraction o f
l eukocytes w ith v ascular g raft m aterials [8]. A s a
r esult o f l eukocyte a dhesion, s everal r eactions m ay
0162-3109/96/$15.00 1996 Elsevier Science B.V. All rights reserved
PII S 0162-3109(96)00003-3
20
L.-Y.L. Yung et al. / Immunopharmacology 32 (1996) 1 9-23
b e i nitiated s uch as l eukocyte s preading, f ormation
o f m icrothrombi t hrough p latelet-platelet a nd
p latelet-leukocyte i nteractions, d etachment o f
t hrombi b y a ction o f l eukocyte p roteases, a nd det achment o f a dherent p latelets a nd a dsorbed p roteins
b y l eukocytes. I n a ddition, r elease o f l eukocyte e nz ymes s uch as e lastase a nd c athepsin G m ay g ive
r ise b oth t o l ocal a nd s ystemic v ascular r eactions o r
t issue d estruction.
A dhesion o f l eukocytes t o s olid s urfaces d epends
o n m any d ifferent f actors t hat i nclude s urface f ree
e nergy [9], s urface h ydrophilicity [10], s urface c hemi stry [ ll], s urface c harge [12], p rotein a dsorption
[13], a nd a dhesion o f o ther c ells s uch as p latelets
[14].
W hile t he r ole o f p lasma p roteins in m ediating
s ubsequent p latelet i nteractions h ave b een w ell c hara cterized, s tudies i nvolving n eutrophil i nteractions
h ave p roduced c onflicting results. W hile s ome h ave
r eported t hat n eutrophils b ehave s imilarly t o p latelets,
o thers h ave s hown n o c orrelation b etween n eutrophil
a dhesion a nd a ctivation a nd t he p resence o f p roteins
p ossessing t he t ripeptide s equence R GD ( Arg, G ly,
A sp), w hich is t he c ell b inding l igand r ecognized b y
p latelets a nd f ibroblasts.
H ere w e a pply a n ovel m ethodology a nd a nalysis
f or q uantitative c haracterization o f n eutrophil a dhes ion t o s urfaces as a f unction o f f luid s hear r ate in a
r adial f low c hamber[15]. T he e ffects o f p readsorbed
h igh-molecular-weight k ininogen ( HK) a nd p reads orbed f ibrinogen o n n eutrophil a dhesion w ere i nvest igated, w ith a b are glass s urface as the c ontrol.
2. Materials a nd m ethodology
2.1. Isolation o f human neutrophils
H uman n eutrophils w ere i solated f rom w hole
b lood a nticoagulated w ith c itrate p hosphate d extrose
u sing a c entrifugation t echnique w ith N eutrophil I sol ation M edium ( NIM TM, C ardinal A ssociates, S anta
Fe, N M) as t he i solation m edium. I n a 15 m l
p olystyrene c entrifuge t ube, 7 m l o f w hole b lood w as
g ently l ayered o ver 4 m l N IM a nd c entrifuged at
4 00 X g f or 3 0 m in a t r oom t emperature. T he P MN
l ayer was t ransferred t o a c lean 5 0 m l t ube, w ashed
w ith H ank's B alanced S alt S olution ( x 1 H BSS, L ife
T echnologies, G aithersburg, M D), a nd c entrifuged at
3 50 g f or 10 m in. T he P MN p ellet was r esusp ended in 10 m l o f e rythrocyte l ysis b uffer ( E-Lysing, C ardinal A ssociates, S anta Fe, N M). T he susp ension was v ortexed a nd c entrifuged at 2 50 X g f or
5 m in, a nd the c ell p ellet was w ashed t wo t imes w ith
H BSS. A fter t he last wash, t he c ell p ellet was r esusp ended in 2 ml o f H BSS c ontaining b ovine s erum
a lbumin ( 2 m g / m l B SA, B axter B ioTech., G lendale,
CA). T he p urity, v iability, a nd c oncentration o f the
c ells w ere e valuated u nder a n o ptical m icroscope
u sing a h emacytometer ( VWR S cientific, B ridgeport,
N J). T he p urity o f t he c ell s uspension was a pproxim ately 9 5-98% as d etermined b y W right's stain.
T he v iability o f c ells in e ach e xperiment was a lways
g reater t han 9 5% as d etermined b y t rypan b lue e xc lusion. T he c oncentration o f c ells was a lways g reater
t han 107 c ells/ml, a nd this was d iluted t o a s tandard
c oncentration o f 105 c ells/ml f or t he a dhesion e xp eriments. T his c oncentration was h igh e nough t o
p rovide g ood v isualization o f c ell a dhesion, b ut dil ute e nough t o m inimize i nteractions b etween c ells
a nd t o m inimize the f ormation o f c ell a ggregates.
2.2. Radial f low apparatus
N eutrophil a dhesion e xperiments w ere p erformed
u sing a r adial f low c hamber w ith a n a utomated v ideo
m icroscopy s ystem.
2.2. I. Radial f low chamber
T he radial f low g eometry p rovides a w ell d efined
l aminar f low f ield w ith a c ontinuous v ariation o f
s hear r ate f or the s tudy o f n eutrophil a dhesion. A
s chematic o f t he r adial f low c hamber u sed in the
e xperiments is s hown in Fig. 1. T he f low c ell was
c onstructed a ccording t o a s imilar d esign b y
C ozens-Roberts e t al. [16]. T he c hamber was m ade
o f a P lexiglas h ousing, w ith t wo o ptically f lat glass
d iscs, o ne in 75 m m a nd the o ther in 5 0 m m in
d iameter. T he 5 0 m m glass disc was p ermanently
a ttached t o the h ousing. T he 75 m m glass disc was
r emovable a nd was p retreated u nder d ifferent c ondit p ions rior t o the a dhesion e xperiment. T he f low c ell
was s ealed u sing a s ystem o f O -rings a nd t ightening
s crews. T he n eutrophil s uspension e ntered at the
c enter o f the c hamber a nd f lowed r adially o utward
b etween the t wo glass d iscs w ith t he gap. T he f luid
L -EL. Y ung et a l./ lmmunopharmacology 32 (1996) 19-23
t hen e xited t hrough t hree e xit p orts t o a w aste c ont ainer.
T he h ydrodynamics o f t he r adial f low c hamber
h ave b een w ell d efined [17]. F or l aminar f low, w here
i nertial t erms a re n eglected, t he v elocity p rofile, u,
is:
Scanned Fields
2.5
2
~'~ 1.5
~
u(r,z)
=
rrrh ~ t 7
( 1)
-
w here Q is v olumetric f low r ate, h is t he g ap w idth,
r is t he r adial d istance f rom t he c enter, a nd z is t he
v ertical p osition f rom t he s urface o f t he g lass d isc.
T he s hear r ate a t t he s urface o f t he g lass d isc, y~, is:
3Q
" L.- n 'rh 2
21
0.5
~
o
Fig. 2. D iagram o f f ields s canned in t he radial f low c hamber.
( 2)
a nd is i nversely p roportional t o r . C ell a dhesion
o ccurs i f t he i nteraction b etween t he c ell a nd t he
s urface o f t he d isc is l arger t han t he s hear f orce
d eveloped b y t he f luid a t t hat p oint o n t he s urface o f
t he d isc.
2.2.2. A utomated uideo microscopy system
T he r adial f low c hamber w as m ounted o n t he
s tage o f a N ikon D iaphot I nverted M icroscope a cc ording t o t he d esign b y D ickinson a nd C ooper [15].
T he s tage o f t he m icroscope h as a m otorized t hree
a xis c ontrol, w hich a llowed r apid a utomated m eas urements o f c ell s urface d ensity a s a f unction o f
t ime a nd p osition in t he c hamber. A s chematic d iag ram o f t he s canned f ields d uring t he e xperiment is
s hown in Fig. 2, w here t he f ields w ith a l inear
d istribution o f s hear r ates, b ut n ot r adii, w ere s canned.
T he i mages w ere o btained u sing a N ewvicon v ideo
c amera m ounted in a s ide p ort o f t he m icroscope a nd
p rocessed a nd r ecorded a t 6 00 m agnification o n a
v ideo c assette u sing t he O ptimas i mage p rocessing
s oftware. T he i mages w ere a nalyzed a fter c ollection
t o o btain t he s urface d ensity o f t he c ells.
2.3. Experimental protocol
2.3.1. Disc preparation
~
/O-Rings
75 turnGlassD i s c . . . . . . . ~ C ~
~
50 mm Glass Disc
P lexiglas ~
Flo~
"
I
Flow
~low
Gap=3001ara
L
Flow
Fig. I. S chematic d iagram o f radial f low c hamber.
T he 75 m m o ptically f lat g lass d iscs w ere c leaned
b y f irst s oaking o vernight in c oncentrated c hromium
t rioxide/sulfuric a cid s olution ( 25 m l M onostat
C hromerge p er 9 lb. c oncentrated s ulfuric a cid, B axt er D iagnostics, M cGaw P ark, I L) t hen s oaking t wice
f or 1 h in t wice d istilled, d eionized w ater ( ddH20),
t wice f or 1 h in m ethanol, a nd d ried o vernight in a n
o ven a t 55C. A ny v isible d ust p articles w ere r em oved u sing a n itrogen g as g un ( Millipore, M arlboro ugh, M A).
D iscs w ere i ncubated o vernight in b uffer b efore
t he e xperiments, a nd t hen p retreated in o ne o f t he
f ollowing w ays: ( 1) 2 h in b uffer a lone a s a b are
c ontrol; ( 2) 2 h i n 10 / z g / m l o f f ibrinogen; a nd ( 3) 2
h in 10 / xg/ml o f h igh-molecular-weight k ininogen.
D uring t reatment, t he d iscs w ere g ently s wirled in
t he p rotein s olutions ( or c ontrol b uffer) o n a r otating
22
L.-Y.L. Yung et a l./ lmmunopharmacology 32 (1996) 1 9-23
2 0se-I
1400
1200
E
E 1000
~
A cknowledgements
:7'S
r,I
T he a uthors a cknowledge f inancial s upport f rom
t he N ational I nstitute o f H ealth t hrough g rants H L4 7179 a nd S pecialized C enter o f R esearch in T hromb osis g rant H L-45486.
~H
8 00
60O
R eferences
~
4 00
2 00
~
! !__I I ! ! ! I I
o:
5
10
15
20
time (rain)
F ig. 3. C hange o f s urface c ell d ensity o n p readsorbed p rotein
s urfaces at s hear rate o f 20 s - ~.
p latform. A fter t he t reatment p eriod, t he p rotein
s olution w as r eplaced w ith b uffer.
2.3.2. N eutrophil a dhesion p rotocol
T he 105 c ells/ml n eutrophil s uspension w as t hen
i ntroduced i nto t he c hamber f or t he a ctual c ell a dhes ion e xperiment w ith a c ontinuous f ield s can o ver
t ime. T he v olumetric f low r ate w as m aintained a t 2.5
m l / m i n . T he d uration o f e ach e xperiment w as a pp rox. 2 0 m in.
3. Results and d iscussion
F ig. 3 s hows t he r esults o f t he c hanges o f s urface
n eutrophil d ensity, C s, w ith t ime, o n d ifferent p reads orbed p rotein s urfaces a t s hear r ate o f 2 0 s - I . T he
s urface c ell d ensity i ncreases a pproximately l inearly
w ith t ime o n a ll s urfaces. T he n umber o f a dherent
c ells o n t he p readsorbed F GN s urface w as s imilar t o
t hat o n t he b are g lass s urface, w hile t he n umber o f
a dherent c ells o n p readsorbed h igh-molecular-weight
k ininogen s urface w as l ess t han 1 / 5 t hat o n t he b are
g lass s urface. I t is c lear t hat s urfaces p readsorbed
w ith H K i nhibit n eutrophil a dhesion c ompared t o
b are g lass a nd s urfaces p readsorbed w ith f ibrinogen,
w hich s upports t he o bservations o f A sabura e t al.
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