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Electrochemical aspects of the neutrophil response to surface-modified polystyrene microspheres
63
Bioelectrochemtstry and Btoenergettcs, 21 (1989) 63-69 A section of ./, £1eetroanal. Chent, and constituting VoL 275 (1989) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
E;ectrocheniieai aspects of the neutrophil response to surface-modified polystyrene microspheres * ,'~._-,o-...~ D. Cut~itta "opt. of BiologF, ,¢.eton Hall University, South O;'ange, NJ 07079 (U.S.A.)
Bruce D ' ]~A1a'low PenKen~ In~, Bedford .Wills, N Y 10507 (U.S.A.)
Vincent A. DeBari ** The RenalLaboratory, St. Joseph's Hospital and Medical Center, Paterson, NJ 07503 (U.S.A.) and £pept. of Medicine, School of Graduate Medical Education, So,on Hall University, South Orange, NJ 07079 (U.S.A.) (Received 10 M a y 1988; in revised form 20 October 1988)
ABSTRACT An empirical study has been made of the relationship between the electrophoretic mobilities (L~) o f polysty,'ene lati~_xs b e a ~ g defined Gurface. functional groups and h u m a n polymorphonuclear neutrophil (PMIq) phagocytic responses. Zeta potentials were calculated from mean values of electrophcretic mobifity distributions (Ue) and correlated with each of four parameters generated from bimodal k:netic curves of luminal-dependent chemiluminescence (CL) as well as with two direct phagocytic indices. Although only one C L parameter and one phagocyte index correspondin:~ ~.o *.he secondary phase of the P M N response correlate significantly, one of vvo phagocytic paramete.-s and all four C L parameters corresponding to the primary mode yielded significant correlations. T~ese data suggest that surface electrochemistry may, in part, be responsible for P M N - p a r t i c l e interactions and that the initial phase of the birr.odal C L response may be due to interracial phenomena.
INTK')DUCTION
A system which m a y be useful in mof2eling cell-surface interactions is the adhesion and phagocytosis of particulates by polymorphonuclear neutrophils (PMN) * Portions of this work were presented at the 32nd Am3u,'d Meeting of the Biophysical So~iety (U.S.A.), Phoenix, AZ, 28 F e b r u a r y - 3 March, 1988 (Abstract: Biophys. J., 53 (1988) 12A). * * T o whom all correspondence should be addresse6 at St. Joseph's Hospital and Medical Center, 703 Main St., P~terson, NJ 07503, U.S.A. 0302-4598/89/$03.50
© 1989 FAsevier Sequoia S.A.
in the absence o f sexum or o t h e r soluble protein sources..These interactions have been modeled in a variety of ways and the. current concept, based o n bioenergetic considerations of the surface [1,2] h a v e led to the und~-staxtding [3,4] .that: (a) the appr0aching cell must generate a radius of curvature oz a ch~racteristzc size ( < 50 rim) and (b) water must be excluded from the interface, i.e., the surface must be hYdr0phobic. The first of these conditions is met b y the ability of the Pl~[]~I to form pseud0Podia, the second condition depends, of course, on the nature of the surface to which the cell is attemptmgz to bhtd. In fact, it has been shown that as the hydrophobicity of the surface of bacteria increases, as assessed by contact angle measurements, the less likely that i~ will be pathogenic [5]. Recently, we reported ~ study of the PI~[H response to polystyrene microspheres having defined surface functional groups [6]. The results of that work indicated that phagocytosis of these particles and the luminol-dcpendent chemiluminescence profiles as indicators of "active-oxygen" species [7] followed a consistent and statistically significant pattern. Underivatized particles provided t h e strongest response, followed by carboxylated, hydroxylated and amino-bearing microsp!teres, in that order. Although the strong response from underivatized polystyrene rnicrospheres was expected from the aforementioned interfacial considerations, the varying degrees of hydrophilic character imparted by - C O O H , - O H and - N H 2 groups o n t h e surface are not directly attributable to hydration effects. ~ To examine the relationship of surface phenomena to PI~fN phagocytosis further, We have subjected the particles in question to electrophoretic measurements. Calculations of the potential at the shear plane (the zeta potential, ~'), suggest that it may be possible to mode| these interactions on the basis of surface electrochemistry. EXPERIMENTAL
P M N harvesting, C L and phagocytosis measurements These have been described pteviousl3, in detail [6,8,9]. The CL and phagocytosis data presented herein were taken from the initial report of this study [6]. Briefly, the four CL pzrameters, generated from bimodal kinetic curves of CL, are: (1) time to peak ( t ) in minutes (min); (2) peak height, i.e., the vzlocitY of photon generation (v) in C P M (counts/rnin); (3) rate of change of velocity with respect to time ( r ) in CPM 2 (coun~s/min2), given by the slope of the ascending portion of the curve, and • 4) total counts, given by the area under the curve ( a ) in counts. Thes¢ are shown schematically in Fig. 1. The two phagocytosis parameters were c, the percent of cells containing at least one particle, and p, the number of particles/ceU ( p / c ) . CL and phagocytosis measurements are indicated for both the primary mode and the secondary mode (indicated by subscripts 1 and 2 respectively). Cell and particle electrophoresis Polystyrene latices were exhaustively dialyzed vs. p H 7.4 hydroxyethylpiperazine ethane sulfonate (10 mM)-buffered Ringer's solution containing 1 m M CaCI (HEPES-buffered Ringer's, HBR). PMN, prepared as described previously [6] were
i
.,
"
, v2 I
//
"
~
'
,
.-.
~
•
;
o.s .=t a2
TIME Fig. i . Schematic representation of C L kinetic plot and derivation ot C L paramete.~s. Peak heights are
given by vt and va, -rate of change of velocity is given as r= and rz (solid line and dashed line, respectively) and half-areas under the curve by a t / 2 and az/2. shown as shaded regions. w a s h e d three times in H B R . S a m p l e s were p r e p a r e d b y placing 25 l=l of 5% ( w / v ) o f particles in 80 ml H B R o r by diluting cells to a final c o n c e n t r a t i o n o f 1 0 6 / m l o f H B R a n d w e r e t h e n aspirated i n t o the c h a m b e r o f a P e n K e m m o d e l 3000 A u t o m a t e d Electrokinetics A n a l y z e r w h i c h h a s b e e n described in detail elsewhere [10]. This i n s t r u m e n t utilizes laser velocimetry to o b t a i n the electrophoretic mobifity distribution. T h e h i s t o g r a m s g e n e r a t e d w e r e stored digitally a n d those p r e s e n t e d r e p r e s e n t the averages o f the individual experiments. Calculations of ~" were b a s e d on the S m o l u c h o w s k i e q u a t i o n [11]:
.¢= u= / 0D
(1)
w h i c h is valid for a n y potential provided the thickness o f the sphere o f c o u n t e r i o n s s u r r o u n d i n g the particle is m u c h smaller t h a n the radius o f the particle. This c o n d i t i o n is invariably m e t in p h y d o l o g i c a l strength saline solutions. In m a k i n g these calculations, t h e :mean value of Ue (Ue), the electrophoretic mobility, was used a n d the t e r m ~ / ( o D (where "8 is viscosity, D is the dielectric c o n s t a n t a n d ¢o is the permittivity o f free space) reduces to a c o n s t a n t for the system such that: ~"ffi 7 , X 1.28 X 10" N . s - V / m C (2) with ~" in V a n d t~dq= 77 in m 2 V - 1 s - 1 .
,Statistical analyses P a r a m e t r i c correlation m;alysis (iJnear regression) w a s p e r f o r m e d using s o f t w a r e f r o m H u m a n S y s t e m s D3ma.ndes Inc., Northridg¢, C A ( P C StafisticianXM). RESULTS
T h e electrophoretic mobility distributions o f P M N are fairly c o n s t a n t as c a n b e seen in Fig. 2. T h e value of ~" r e p o r t e d previously for P M N s u s p e n d e d in 0.145 M
Y
66
_~
:, _~
,
10- s U e / m z V - i s - t
Fig. 2. Histograms o f electrophoretic mobility in H B R of P M N from three representative subjects, given as computer-averaged mean ( n ==6 for .~ch subject) distributions.
N a C I is - - 1 2 m V [3], a value n o t a p p r e c i a b l y d i f f e r e n t f r o m the m e a n v a l u e o f - 1 1 . 2 m V which we d e t e r m i n e d for cells s u s p e n d e d in H B R , P r e s e n t e d in Fig. 3 a r e the m e a n d i s t r i b u t i o n hist(~gram for t h e f o u r types o f m~erospheres we studied. T h e resu)th~g m e a n s a n d s t a n d a r d e r r o r s for the ~" d e ~ v e d f r o m t h e electrophoretic d a t a are p r e s e n t e d in T a b l e 1. T h e c h a r g e o n the trader-
I0 -eue,/m~ v's-1 Fig. 3. Computed m e a n electrc~horetic mobilities, Ue, for, from top to bottom, under/vatized ( ~ == - 3.459×10 - s m2/V s), carboxy (U0 = - - 3 . 3 5 9 × I 0 - S m 2 / V s), hydroxy (Ue = - 2 . 9 8 1 × I 0 - s m 2 / V s) and amino (U~ = - - 2 . 2 9 3 × I 0 - s m2/V s) microspheres. Each histogram is the averaged result o f six measurements.
TABLE1
.~ • • ' .
•V a l u e s o f ~' d e r i v e d f r o m ~ele~lrophoretic a n a l y s e s . D a t a e x p r c s s ~
experiment
"
.
Particle
Hydroxyl (10 Amino (A)
as me, ms4-1 SEM; :
r
"
'
n =6
f o r e a c h ." "
~/mV
Underivatized (U)
Carboxyl ( ~
'
- 4 4 . 3 ~ 2.036 .
- 43.0+ 0.012 -- 38.2 d : 0 . 3 3 3 -- 2 9 . 4 4- 0 . 6 3 4
'
ivatized particles, comparable to the carboxylated latex, is, most likely, due to sulfate groups covalently attached to the surface which result from the use o f persulfate initiaU>rs in the free-radical polymerization process used to manufacture these particles [12]. ])epending on the prior history of the latices, the sulfate groups can be hydrolyzed to produce an hydroxylated surface [13]. The buffer system itself also has a profound effect on the nature of the ionic double layer and, ultimately, the observed value of g'. There is ample evidence for the variation of ~" with p H and ionic strength and composition; in pilot experiments using saline suspensions of these rnicrospheres we observed values of ~" ranging from --2I~ mV for the underivatized particles to 4-1 mV for the amino microspheres. The near electroneutrality of ~" for the amino particles in unbuffered aqueous suspension is; in fact, reflected in their strong tendency to flocculate. Regression analyses of the phagocytic and CL parameters are presented in Table 2. With the exception of time, all other parameters demonstrate an inverse relationship with ~'. Using the 95% confidence limits ( p ~<0.05) to assess significance, one can observe that for the primary mode parameters, all but c I cotTelate significantly with ~; of the secondary mode parameters, the correlations of only one CL parameter and one phagocytic index achieved statistical siEnificance. DISCUSSION
The P M N is a highly specialiTed cell which has evolved primarily to interact with microorganisms invading the mammalian body. The bactericidal mechanisms responsible for this important host-defense activity also render the P M N a critical actor in the inflammatory response [14]. Although the P M N responds to a variety of soluble stimuli [15,16], the generation of "active oxygen" reactants (as assessed by CL) may be enhanced in the presence of some particulate stimuli [17,18]. The surface-dependent nature of P M N activation is a well.known phenomenon, but one which is not enti~:ly understood. Surface potentials are an important aspect of colloidal systems, yet, Correlation of these potentials with P M N activity have, in the past, not been fruitful [19]. This may stem partially from the effect of the complexity of naturally occurring surfaces, resulting in highly variable responses to microorganisms [20]. In this study, we have continued the use of a more straightforward model [6] to correlate the potential at
68 TABLE
2
S u m m a r y o f c o r r e l a t i o n d a t a f o r ~" v e r s u s p r i m a r y a n d s e c o n d a r y p h a s e p a r a m e t e r s o f t h e C L r e s p o n s e and phnsocytosis. Slope and intercept of regression equation are given in the second and third columns; s t a t i s t i c a l a n a l y s e s a r e prer, c n t e d i n t h e l a s t t h r e e c o l u m n s Phase parameter
Equation parameter m
al t2
u! V2
al d2 rl fT. ca
¢2 Pl
P2
- -
- -
- -
- -
"
b
0.019 0.139 16.60 21.65 -- 6.37 64.84 17.85 -- 6,72 - - 1.28 1.44 -- 0.196 --0.244 - -
1.78 9.54 -- 433.94 -- 530.56 - - 135.21 - - 15'31.83 -- 469.93 -- 178.13 17.90 16.42 - - 1.32 --1.23
Statistical data b r
0.986 0.948 0.983 0.891 0.977 0.858 0.985 0.932 0.939 0.968 0.983 0.877
t
p
8.44 4.21 -- 7.49 -- 2.79 -- 6.48 - - 2.36 - - 8.13 - - 3.65 - - 3.87 - - 5.48 -- 7.52 --2.58
0.011 0.050 0.014 0.108 0.019 0.143 0.012 0.066 0.059 0.028 0.014 0.123
• E q u a t i o n i n t h e f o r m : p a r a m e t e r ( C L o r p h a g o c y t o s i s ) •ffi m~" 4- b. b r is t h e P e a r s o n c o r r e l a t i o n c o e f f i c i e n t , t v a l u e ( S t u d e n t ' s ) i s d e r i v e d f r o m r , a n d p is t h e p r o b a b i f i t y .
t h e s h e a r plane, ~', with biochemical, i.e., C L a n d microscopic m a r k e r s o f P M • action. T h e f i n d i n g o f a relationship b e t w e e n ~" a n d the initial m o d e of P M N a c t i o n is intrigaing. T h e b i m o d a l n a t u r e o f the C L response itself is not fully understood, b u t c a n b e s h o w n to b e d e p e n d e n t , in part, o n the relative strength o f the C L xesponse. T h e strong correlation b e t w e e n the p r i m a r y m o d e pm'ameters a n d ~" is good p r e s u m p t i v e evidence for the r a t h e r intuitive expectation that contact p h e n o m e n a are responsible for this initial ste.g¢ of P M N action. It is t e m p t i n g to speculate o n t h e m e c h a n i s m b y which those particles with a m o r e negative surface potential p r o m o t e a greater response t h a n those with a less negative ~'. I n terms o f signal t r a n s d u c t i o n i n the P M N , it is n o w well-established that C a 2+ m o b i l i z a t i o n is a n i m p o r t a n t m e m b r a n e - m e d i a t e d e.ve.nt [21,221. I n fact, the current concept of a d h e s i o n b a s e d o n attractive V a n clef W a l l s forces [23] avoids the i m p l i c i t physical rigidity o f the older Ca2+-bridge m o d e l [24]. In terms o f our data, it does not seem tmllkely that the a p p r c a c h of a~ incrvasL~gly negative surface c h a r g e could trigger cellular fluxes o f positive, m o n o v a l e n t ionic species, e.g., N a +, K + a n d / o r protons, b r i n g i n g a b o u t a localized d e p o l a r i z a t i o n or h y p e r p o l a r i z a t i o n l i n k e d to a C a 2 . flux. There is, i n fact, s o m e i n d i c a t i o n that az~.fidinated particles, w i t h a strongly positive surface charge (~" ffi -!-28.3 lnV in H a n k s buffer, p H 7.4), i n d u c e superoxide radical g e n e r a t i o n f r o m n e u t r o p h i l s n e a r l y as well as d o sulfated particles [25]. T h i s m i g h t h e l p to explain the a p p a r e n t l y d i s c r e p a n t f i n d i n g s [7] r e g a r d i n g the direction o f the c h a n g e in t r a n s m e m b r a n e p o t e n t i a l w h i c h a c c o m p a nies the activation o f oxygen free radicals i n P M N . T h i s hypothesis, o f course, requires further e x p e r i m e n t a l verification.
'-~
.
.
In conclusion, we suggest that the electrochemical properties of particle surfaces m a y serve to mediate P M N particle interactions and that the primary m o d e of the P M N r e s p o n s e m a y be a ~('unction of interracial phenomena. ACKNOWLEDGEMENTS
We wish to thank the staff at Pandex Laboratorif~s, Mundelein, IL, for the kind gift of the latex particles and especially Dr. Jeff Wang for his helpful discussions. Tony Ingenito provided expert technical assistance in the early phase of this project. Special thanks are due to the management of PenKem, Inc,, for graciously allowing us the use of the System 3000 for electrophoretic distribution measurements. REFERENCES 1 B. I~rjaguin and L.D. L a n d a , , Acta Physicochim. URSS, 14 (1941) 633. 2 E.J.N. Vervey and J.T.G. Overbeek, Theory of the Stabifity o f Lyophobic Colloids, Elsevier, Amsterdam, 1948. 3 C J . Van Oss, R J . G o o d and A.W. N e u m a n n , J. Electroanal. Chem., 37 (1972) 387. 4 D.R. A b s o l o m in (3. DiSabato and J. Everse (Eds.). Methods in Enzymology, Vol. 132, Immunochemical Techniq,,es, part J, Phagocytosis and Cell-mediated Cytotoxicity, Academic Press, Orlando, FL, 1986, p. 65. 5 C J . Van Oss and C.F. Gillman, J. Reticuloendothelial Soc., 12 (1972) 283. 6 A.$. Orsird, A.C. ingenlto, M.A. Needle and V.A. DeBari, Cell Biophys., 10 (1937) 33. 7 B.M. Babior, .I. Clin. Invest., "/3 (1984) 599. 8 V.A. DeBari, P.M. Uychich, A_w. Orsini, A.C. Ingenito and M.A. Needle, Trans. Am. So(:. ArtiL Intern. Organs, 32 (1936) 597. 9 V.A. DeBari, O. Frank, H. Baker and M.A. Needle, Am. J. Clin. Nutr., 39 (1984) 410. 10 P J . G o e t z in W. Sch,ztt a;ld H. Ktinkman (Eds.), C_~dl Electrophoresis, Walter D e G r u y t e r and Co., Berlin, 1985, p. 41. I1 R J . Hunter, Zeta Potentizds and Surface Chemistry, Academic Press, N e w York, 19;H, p. 59. 12 L.B. Bangs, Uniform Latex Particles, Seragen Diagnostics, Indianapolis, IN, 1985, p. 5. 13 H J . Van de Hul and J.W. Van der Hoff, Brit. Polym. J., 2 (1970) 121. 14 J.L Gallin, Clin. Rcs., 32 (1984) 320. 15 D. Romeo, Trends Biochem. Sci., 7 (1982) 408. 16 P.H. Naccache, M.M. Molski, M. Volpi. J. S~:hefcyk, T.F.P. Molski, L. L o w , E.L. Becker and R.I. Sha'afi, J. Leukocyte Biol., 40 (1986) 533. 17 J. Nicotra, A J . Orsini and V.A. DeBari, Cell Biophys., 7 (1985) 283. 18 S.V. Shah, J.D. Wa]lin and S.D. Eilen, .L Clin. Endocrinol. Metab., 57 (1983) 402. 19 C J . Van Oss, D.R. Absotom and A.W. N e u m a n n in S.NI. Reichard and $.P. Filkins (Eds.), 'File Reticyloendothelial System, Vol. 7A, Plenum, 1984, p. 23. 20 P. Robinson, D. Wakefield, S.N. Breit, J.F. Easter and R. Penny, Infect. immun., 43 (1984) 744. 21 J.R. White, P.H. Nacc.aehe, T.F.P. Molski, P. Borgeat and R.I. Sha'afi, Biochem. Biophys. Rcs. Conunun., 113 (1983) 44. 22 R. Snyderman, C.D. Smith and M.W. Veighese, $. L~ukocyte Biol., 40 (1986) 785. 23 A.W. N~umann, 3.N. Omenyi and C J . ~ran Oss, Colloid Polym. Sci., 257 (1979) 413. 24 A.D. Bangbam, Ann. N.Y. Acad. Sci., 116 (1964) 945. 25 D.A. Berretta, N.V. Resureccion, D.G. Baker, J.S. Bomalski and H.R. Schumacher, Clin. Res., 36 (1988) 530A (abstract).
Bioelectrochemtstry and Btoenergettcs, 21 (1989) 63-69 A section of ./, £1eetroanal. Chent, and constituting VoL 275 (1989) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
E;ectrocheniieai aspects of the neutrophil response to surface-modified polystyrene microspheres * ,'~._-,o-...~ D. Cut~itta "opt. of BiologF, ,¢.eton Hall University, South O;'ange, NJ 07079 (U.S.A.)
Bruce D ' ]~A1a'low PenKen~ In~, Bedford .Wills, N Y 10507 (U.S.A.)
Vincent A. DeBari ** The RenalLaboratory, St. Joseph's Hospital and Medical Center, Paterson, NJ 07503 (U.S.A.) and £pept. of Medicine, School of Graduate Medical Education, So,on Hall University, South Orange, NJ 07079 (U.S.A.) (Received 10 M a y 1988; in revised form 20 October 1988)
ABSTRACT An empirical study has been made of the relationship between the electrophoretic mobilities (L~) o f polysty,'ene lati~_xs b e a ~ g defined Gurface. functional groups and h u m a n polymorphonuclear neutrophil (PMIq) phagocytic responses. Zeta potentials were calculated from mean values of electrophcretic mobifity distributions (Ue) and correlated with each of four parameters generated from bimodal k:netic curves of luminal-dependent chemiluminescence (CL) as well as with two direct phagocytic indices. Although only one C L parameter and one phagocyte index correspondin:~ ~.o *.he secondary phase of the P M N response correlate significantly, one of vvo phagocytic paramete.-s and all four C L parameters corresponding to the primary mode yielded significant correlations. T~ese data suggest that surface electrochemistry may, in part, be responsible for P M N - p a r t i c l e interactions and that the initial phase of the birr.odal C L response may be due to interracial phenomena.
INTK')DUCTION
A system which m a y be useful in mof2eling cell-surface interactions is the adhesion and phagocytosis of particulates by polymorphonuclear neutrophils (PMN) * Portions of this work were presented at the 32nd Am3u,'d Meeting of the Biophysical So~iety (U.S.A.), Phoenix, AZ, 28 F e b r u a r y - 3 March, 1988 (Abstract: Biophys. J., 53 (1988) 12A). * * T o whom all correspondence should be addresse6 at St. Joseph's Hospital and Medical Center, 703 Main St., P~terson, NJ 07503, U.S.A. 0302-4598/89/$03.50
© 1989 FAsevier Sequoia S.A.
in the absence o f sexum or o t h e r soluble protein sources..These interactions have been modeled in a variety of ways and the. current concept, based o n bioenergetic considerations of the surface [1,2] h a v e led to the und~-staxtding [3,4] .that: (a) the appr0aching cell must generate a radius of curvature oz a ch~racteristzc size ( < 50 rim) and (b) water must be excluded from the interface, i.e., the surface must be hYdr0phobic. The first of these conditions is met b y the ability of the Pl~[]~I to form pseud0Podia, the second condition depends, of course, on the nature of the surface to which the cell is attemptmgz to bhtd. In fact, it has been shown that as the hydrophobicity of the surface of bacteria increases, as assessed by contact angle measurements, the less likely that i~ will be pathogenic [5]. Recently, we reported ~ study of the PI~[H response to polystyrene microspheres having defined surface functional groups [6]. The results of that work indicated that phagocytosis of these particles and the luminol-dcpendent chemiluminescence profiles as indicators of "active-oxygen" species [7] followed a consistent and statistically significant pattern. Underivatized particles provided t h e strongest response, followed by carboxylated, hydroxylated and amino-bearing microsp!teres, in that order. Although the strong response from underivatized polystyrene rnicrospheres was expected from the aforementioned interfacial considerations, the varying degrees of hydrophilic character imparted by - C O O H , - O H and - N H 2 groups o n t h e surface are not directly attributable to hydration effects. ~ To examine the relationship of surface phenomena to PI~fN phagocytosis further, We have subjected the particles in question to electrophoretic measurements. Calculations of the potential at the shear plane (the zeta potential, ~'), suggest that it may be possible to mode| these interactions on the basis of surface electrochemistry. EXPERIMENTAL
P M N harvesting, C L and phagocytosis measurements These have been described pteviousl3, in detail [6,8,9]. The CL and phagocytosis data presented herein were taken from the initial report of this study [6]. Briefly, the four CL pzrameters, generated from bimodal kinetic curves of CL, are: (1) time to peak ( t ) in minutes (min); (2) peak height, i.e., the vzlocitY of photon generation (v) in C P M (counts/rnin); (3) rate of change of velocity with respect to time ( r ) in CPM 2 (coun~s/min2), given by the slope of the ascending portion of the curve, and • 4) total counts, given by the area under the curve ( a ) in counts. Thes¢ are shown schematically in Fig. 1. The two phagocytosis parameters were c, the percent of cells containing at least one particle, and p, the number of particles/ceU ( p / c ) . CL and phagocytosis measurements are indicated for both the primary mode and the secondary mode (indicated by subscripts 1 and 2 respectively). Cell and particle electrophoresis Polystyrene latices were exhaustively dialyzed vs. p H 7.4 hydroxyethylpiperazine ethane sulfonate (10 mM)-buffered Ringer's solution containing 1 m M CaCI (HEPES-buffered Ringer's, HBR). PMN, prepared as described previously [6] were
i
.,
"
, v2 I
//
"
~
'
,
.-.
~
•
;
o.s .=t a2
TIME Fig. i . Schematic representation of C L kinetic plot and derivation ot C L paramete.~s. Peak heights are
given by vt and va, -rate of change of velocity is given as r= and rz (solid line and dashed line, respectively) and half-areas under the curve by a t / 2 and az/2. shown as shaded regions. w a s h e d three times in H B R . S a m p l e s were p r e p a r e d b y placing 25 l=l of 5% ( w / v ) o f particles in 80 ml H B R o r by diluting cells to a final c o n c e n t r a t i o n o f 1 0 6 / m l o f H B R a n d w e r e t h e n aspirated i n t o the c h a m b e r o f a P e n K e m m o d e l 3000 A u t o m a t e d Electrokinetics A n a l y z e r w h i c h h a s b e e n described in detail elsewhere [10]. This i n s t r u m e n t utilizes laser velocimetry to o b t a i n the electrophoretic mobifity distribution. T h e h i s t o g r a m s g e n e r a t e d w e r e stored digitally a n d those p r e s e n t e d r e p r e s e n t the averages o f the individual experiments. Calculations of ~" were b a s e d on the S m o l u c h o w s k i e q u a t i o n [11]:
.¢= u= / 0D
(1)
w h i c h is valid for a n y potential provided the thickness o f the sphere o f c o u n t e r i o n s s u r r o u n d i n g the particle is m u c h smaller t h a n the radius o f the particle. This c o n d i t i o n is invariably m e t in p h y d o l o g i c a l strength saline solutions. In m a k i n g these calculations, t h e :mean value of Ue (Ue), the electrophoretic mobility, was used a n d the t e r m ~ / ( o D (where "8 is viscosity, D is the dielectric c o n s t a n t a n d ¢o is the permittivity o f free space) reduces to a c o n s t a n t for the system such that: ~"ffi 7 , X 1.28 X 10" N . s - V / m C (2) with ~" in V a n d t~dq= 77 in m 2 V - 1 s - 1 .
,Statistical analyses P a r a m e t r i c correlation m;alysis (iJnear regression) w a s p e r f o r m e d using s o f t w a r e f r o m H u m a n S y s t e m s D3ma.ndes Inc., Northridg¢, C A ( P C StafisticianXM). RESULTS
T h e electrophoretic mobility distributions o f P M N are fairly c o n s t a n t as c a n b e seen in Fig. 2. T h e value of ~" r e p o r t e d previously for P M N s u s p e n d e d in 0.145 M
Y
66
_~
:, _~
,
10- s U e / m z V - i s - t
Fig. 2. Histograms o f electrophoretic mobility in H B R of P M N from three representative subjects, given as computer-averaged mean ( n ==6 for .~ch subject) distributions.
N a C I is - - 1 2 m V [3], a value n o t a p p r e c i a b l y d i f f e r e n t f r o m the m e a n v a l u e o f - 1 1 . 2 m V which we d e t e r m i n e d for cells s u s p e n d e d in H B R , P r e s e n t e d in Fig. 3 a r e the m e a n d i s t r i b u t i o n hist(~gram for t h e f o u r types o f m~erospheres we studied. T h e resu)th~g m e a n s a n d s t a n d a r d e r r o r s for the ~" d e ~ v e d f r o m t h e electrophoretic d a t a are p r e s e n t e d in T a b l e 1. T h e c h a r g e o n the trader-
I0 -eue,/m~ v's-1 Fig. 3. Computed m e a n electrc~horetic mobilities, Ue, for, from top to bottom, under/vatized ( ~ == - 3.459×10 - s m2/V s), carboxy (U0 = - - 3 . 3 5 9 × I 0 - S m 2 / V s), hydroxy (Ue = - 2 . 9 8 1 × I 0 - s m 2 / V s) and amino (U~ = - - 2 . 2 9 3 × I 0 - s m2/V s) microspheres. Each histogram is the averaged result o f six measurements.
TABLE1
.~ • • ' .
•V a l u e s o f ~' d e r i v e d f r o m ~ele~lrophoretic a n a l y s e s . D a t a e x p r c s s ~
experiment
"
.
Particle
Hydroxyl (10 Amino (A)
as me, ms4-1 SEM; :
r
"
'
n =6
f o r e a c h ." "
~/mV
Underivatized (U)
Carboxyl ( ~
'
- 4 4 . 3 ~ 2.036 .
- 43.0+ 0.012 -- 38.2 d : 0 . 3 3 3 -- 2 9 . 4 4- 0 . 6 3 4
'
ivatized particles, comparable to the carboxylated latex, is, most likely, due to sulfate groups covalently attached to the surface which result from the use o f persulfate initiaU>rs in the free-radical polymerization process used to manufacture these particles [12]. ])epending on the prior history of the latices, the sulfate groups can be hydrolyzed to produce an hydroxylated surface [13]. The buffer system itself also has a profound effect on the nature of the ionic double layer and, ultimately, the observed value of g'. There is ample evidence for the variation of ~" with p H and ionic strength and composition; in pilot experiments using saline suspensions of these rnicrospheres we observed values of ~" ranging from --2I~ mV for the underivatized particles to 4-1 mV for the amino microspheres. The near electroneutrality of ~" for the amino particles in unbuffered aqueous suspension is; in fact, reflected in their strong tendency to flocculate. Regression analyses of the phagocytic and CL parameters are presented in Table 2. With the exception of time, all other parameters demonstrate an inverse relationship with ~'. Using the 95% confidence limits ( p ~<0.05) to assess significance, one can observe that for the primary mode parameters, all but c I cotTelate significantly with ~; of the secondary mode parameters, the correlations of only one CL parameter and one phagocytic index achieved statistical siEnificance. DISCUSSION
The P M N is a highly specialiTed cell which has evolved primarily to interact with microorganisms invading the mammalian body. The bactericidal mechanisms responsible for this important host-defense activity also render the P M N a critical actor in the inflammatory response [14]. Although the P M N responds to a variety of soluble stimuli [15,16], the generation of "active oxygen" reactants (as assessed by CL) may be enhanced in the presence of some particulate stimuli [17,18]. The surface-dependent nature of P M N activation is a well.known phenomenon, but one which is not enti~:ly understood. Surface potentials are an important aspect of colloidal systems, yet, Correlation of these potentials with P M N activity have, in the past, not been fruitful [19]. This may stem partially from the effect of the complexity of naturally occurring surfaces, resulting in highly variable responses to microorganisms [20]. In this study, we have continued the use of a more straightforward model [6] to correlate the potential at
68 TABLE
2
S u m m a r y o f c o r r e l a t i o n d a t a f o r ~" v e r s u s p r i m a r y a n d s e c o n d a r y p h a s e p a r a m e t e r s o f t h e C L r e s p o n s e and phnsocytosis. Slope and intercept of regression equation are given in the second and third columns; s t a t i s t i c a l a n a l y s e s a r e prer, c n t e d i n t h e l a s t t h r e e c o l u m n s Phase parameter
Equation parameter m
al t2
u! V2
al d2 rl fT. ca
¢2 Pl
P2
- -
- -
- -
- -
"
b
0.019 0.139 16.60 21.65 -- 6.37 64.84 17.85 -- 6,72 - - 1.28 1.44 -- 0.196 --0.244 - -
1.78 9.54 -- 433.94 -- 530.56 - - 135.21 - - 15'31.83 -- 469.93 -- 178.13 17.90 16.42 - - 1.32 --1.23
Statistical data b r
0.986 0.948 0.983 0.891 0.977 0.858 0.985 0.932 0.939 0.968 0.983 0.877
t
p
8.44 4.21 -- 7.49 -- 2.79 -- 6.48 - - 2.36 - - 8.13 - - 3.65 - - 3.87 - - 5.48 -- 7.52 --2.58
0.011 0.050 0.014 0.108 0.019 0.143 0.012 0.066 0.059 0.028 0.014 0.123
• E q u a t i o n i n t h e f o r m : p a r a m e t e r ( C L o r p h a g o c y t o s i s ) •ffi m~" 4- b. b r is t h e P e a r s o n c o r r e l a t i o n c o e f f i c i e n t , t v a l u e ( S t u d e n t ' s ) i s d e r i v e d f r o m r , a n d p is t h e p r o b a b i f i t y .
t h e s h e a r plane, ~', with biochemical, i.e., C L a n d microscopic m a r k e r s o f P M • action. T h e f i n d i n g o f a relationship b e t w e e n ~" a n d the initial m o d e of P M N a c t i o n is intrigaing. T h e b i m o d a l n a t u r e o f the C L response itself is not fully understood, b u t c a n b e s h o w n to b e d e p e n d e n t , in part, o n the relative strength o f the C L xesponse. T h e strong correlation b e t w e e n the p r i m a r y m o d e pm'ameters a n d ~" is good p r e s u m p t i v e evidence for the r a t h e r intuitive expectation that contact p h e n o m e n a are responsible for this initial ste.g¢ of P M N action. It is t e m p t i n g to speculate o n t h e m e c h a n i s m b y which those particles with a m o r e negative surface potential p r o m o t e a greater response t h a n those with a less negative ~'. I n terms o f signal t r a n s d u c t i o n i n the P M N , it is n o w well-established that C a 2+ m o b i l i z a t i o n is a n i m p o r t a n t m e m b r a n e - m e d i a t e d e.ve.nt [21,221. I n fact, the current concept of a d h e s i o n b a s e d o n attractive V a n clef W a l l s forces [23] avoids the i m p l i c i t physical rigidity o f the older Ca2+-bridge m o d e l [24]. In terms o f our data, it does not seem tmllkely that the a p p r c a c h of a~ incrvasL~gly negative surface c h a r g e could trigger cellular fluxes o f positive, m o n o v a l e n t ionic species, e.g., N a +, K + a n d / o r protons, b r i n g i n g a b o u t a localized d e p o l a r i z a t i o n or h y p e r p o l a r i z a t i o n l i n k e d to a C a 2 . flux. There is, i n fact, s o m e i n d i c a t i o n that az~.fidinated particles, w i t h a strongly positive surface charge (~" ffi -!-28.3 lnV in H a n k s buffer, p H 7.4), i n d u c e superoxide radical g e n e r a t i o n f r o m n e u t r o p h i l s n e a r l y as well as d o sulfated particles [25]. T h i s m i g h t h e l p to explain the a p p a r e n t l y d i s c r e p a n t f i n d i n g s [7] r e g a r d i n g the direction o f the c h a n g e in t r a n s m e m b r a n e p o t e n t i a l w h i c h a c c o m p a nies the activation o f oxygen free radicals i n P M N . T h i s hypothesis, o f course, requires further e x p e r i m e n t a l verification.
'-~
.
.
In conclusion, we suggest that the electrochemical properties of particle surfaces m a y serve to mediate P M N particle interactions and that the primary m o d e of the P M N r e s p o n s e m a y be a ~('unction of interracial phenomena. ACKNOWLEDGEMENTS
We wish to thank the staff at Pandex Laboratorif~s, Mundelein, IL, for the kind gift of the latex particles and especially Dr. Jeff Wang for his helpful discussions. Tony Ingenito provided expert technical assistance in the early phase of this project. Special thanks are due to the management of PenKem, Inc,, for graciously allowing us the use of the System 3000 for electrophoretic distribution measurements. REFERENCES 1 B. I~rjaguin and L.D. L a n d a , , Acta Physicochim. URSS, 14 (1941) 633. 2 E.J.N. Vervey and J.T.G. Overbeek, Theory of the Stabifity o f Lyophobic Colloids, Elsevier, Amsterdam, 1948. 3 C J . Van Oss, R J . G o o d and A.W. N e u m a n n , J. Electroanal. Chem., 37 (1972) 387. 4 D.R. A b s o l o m in (3. DiSabato and J. Everse (Eds.). Methods in Enzymology, Vol. 132, Immunochemical Techniq,,es, part J, Phagocytosis and Cell-mediated Cytotoxicity, Academic Press, Orlando, FL, 1986, p. 65. 5 C J . Van Oss and C.F. Gillman, J. Reticuloendothelial Soc., 12 (1972) 283. 6 A.$. Orsird, A.C. ingenlto, M.A. Needle and V.A. DeBari, Cell Biophys., 10 (1937) 33. 7 B.M. Babior, .I. Clin. Invest., "/3 (1984) 599. 8 V.A. DeBari, P.M. Uychich, A_w. Orsini, A.C. Ingenito and M.A. Needle, Trans. Am. So(:. ArtiL Intern. Organs, 32 (1936) 597. 9 V.A. DeBari, O. Frank, H. Baker and M.A. Needle, Am. J. Clin. Nutr., 39 (1984) 410. 10 P J . G o e t z in W. Sch,ztt a;ld H. Ktinkman (Eds.), C_~dl Electrophoresis, Walter D e G r u y t e r and Co., Berlin, 1985, p. 41. I1 R J . Hunter, Zeta Potentizds and Surface Chemistry, Academic Press, N e w York, 19;H, p. 59. 12 L.B. Bangs, Uniform Latex Particles, Seragen Diagnostics, Indianapolis, IN, 1985, p. 5. 13 H J . Van de Hul and J.W. Van der Hoff, Brit. Polym. J., 2 (1970) 121. 14 J.L Gallin, Clin. Rcs., 32 (1984) 320. 15 D. Romeo, Trends Biochem. Sci., 7 (1982) 408. 16 P.H. Naccache, M.M. Molski, M. Volpi. J. S~:hefcyk, T.F.P. Molski, L. L o w , E.L. Becker and R.I. Sha'afi, J. Leukocyte Biol., 40 (1986) 533. 17 J. Nicotra, A J . Orsini and V.A. DeBari, Cell Biophys., 7 (1985) 283. 18 S.V. Shah, J.D. Wa]lin and S.D. Eilen, .L Clin. Endocrinol. Metab., 57 (1983) 402. 19 C J . Van Oss, D.R. Absotom and A.W. N e u m a n n in S.NI. Reichard and $.P. Filkins (Eds.), 'File Reticyloendothelial System, Vol. 7A, Plenum, 1984, p. 23. 20 P. Robinson, D. Wakefield, S.N. Breit, J.F. Easter and R. Penny, Infect. immun., 43 (1984) 744. 21 J.R. White, P.H. Nacc.aehe, T.F.P. Molski, P. Borgeat and R.I. Sha'afi, Biochem. Biophys. Rcs. Conunun., 113 (1983) 44. 22 R. Snyderman, C.D. Smith and M.W. Veighese, $. L~ukocyte Biol., 40 (1986) 785. 23 A.W. N~umann, 3.N. Omenyi and C J . ~ran Oss, Colloid Polym. Sci., 257 (1979) 413. 24 A.D. Bangbam, Ann. N.Y. Acad. Sci., 116 (1964) 945. 25 D.A. Berretta, N.V. Resureccion, D.G. Baker, J.S. Bomalski and H.R. Schumacher, Clin. Res., 36 (1988) 530A (abstract).