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Phagocytosis and hydrophobicity: A method of calculating contact angles based on the diameter of sessile drops
Journal of Immunological Methods, 40 (1981) 171--179 © Elsevier/North-Holland Biomedical Press
171
PHAGOCYTOSIS AND HYDROPHOBICITY: A METHOD OF C A L C U L A T I N G CONTACT A N G L E S BASED ON THE D I A M E T E R OF SESSILE DROPS
C. DAHLGREN and T. SUNQVIST Department of Medical Microbiology, The University of Link6ping, S-581 85 Link6ping, Sweden
(Received 12 May 1980, accepted 1 September 1980)
The correlation between the contact angle and degree of phagocytosis of different yeast particles has been investigated. To facilitate the estimation of the contact angle, we have tested the hypothesis that the shape of a small liquid drop put on a flat surface is that of a truncated sphere. By making this approximation it is possible to calculate the contact angle, i.e. the tangent to the drop in the 3-phase liquid/solid/air meeting point, by measuring the drop diameter. Known volumes of saline were put on different surfaces and the diameters of the drops were measured from above. Calculation of the contact angle with drops of different volumes, and comparison between expected and measured height of 10 pl drops, indicated that the assumption that the shape of a drop is that of a truncated sphere is valid. Monolayers of leukocytes was shown to give rise to a contact angle of 17.9 °. Particles with a lower contact angle than the phagocytic cells resisted phagocytosis, but opsonization of the particles with normal human serum rendered them susceptible to phagocytosis, conferring a higher contact angle than that of the phagocytic cells.
INTRODUCTION The process o f p h a g o c y t o s i s is initiated b y a t t a c h m e n t o f t h e p r e y t o t h e surface o f t h e p h a g o c y t i c cell, f o l l o w e d b y its e n g u l f m e n t and d e g r a d a t i o n . S t u d y o f the p h y s i c o - c h e m i c a l surface p r o p e r t i e s of bacteria in relation to their liability o f p h a g o c y t o s i s has s h o w n t h a t the t e n d e n c y t o h y d r o p h o b i c i n t e r a c t i o n is o f i m p o r t a n c e f o r host-parasite i n t e r a c t i o n (Van Oss et al., 1 9 7 5 ; S t j e r n s t r S m et al., 1 9 7 7 ; M a g n u s s o n et al., 1 9 7 9 ) and for virulence o f t h e b a c t e r i a (Van Oss and Gillman, 1 9 7 2 ; Tagesson and Stendahl, 1973}. D i f f e r e n t m e t h o d s o f e s t i m a t i n g h y d r o p h o b i c i t y have b e e n used t o correlate this w i t h the virulence o f m i c r o o r g a n i s m s (Magnusson et al., 1 9 7 7 ; S t j e r n s t r S m et al., 1 9 7 7 ; V a n Oss, 1 9 7 8 ) . T h e c o n t a c t angle, i.e., the angle f o r m e d b e t w e e n a sessile d r o p o f saline and a flat surface c a r r y i n g a m o n o l a y e r o f cells, has been used as a m e a s u r e o f surface h y d r o p h o b i c i t y (Van Oss and Gillman., 1 9 7 2 ; V a n Oss et al., 1 9 7 5 ; V a n Oss, 1 9 7 8 ) . C o n t a c t angles are usually m e a s u r e d w i t h t h e aid o f a t e l e s c o p e w i t h Crossed hairs
172
attached to a goniometer. In order to simplify the measurement of contact angles, we have tested the approximation that the shape of a drop of saline on a flat surface is that of a truncated sphere, and found that the diameter of a drop of saline on a flat surface can be used to calculate the contact angle. MATERIAL
AND METHODS
Contact angle measurements Known volumes (V) of saline were placed on different surfaces and the diameters of the drops were measured from above with a microscope equipped with an ocular micrometer (Zeiss stereomicroscope with a zoom system). It was hypothesized that the shape of the drop may be approximated to that of a truncated sphere. The volume (V) of a truncated sphere (Standard Mathematical Tables) is
=g~ h ( 3 a 2 + h 2)
V
(1)
where the symbols correspond to those in Fig. 1. The height may be described as
h
a
a
sin 0
tg 0
-
(1.2)
and a substitution of equation 1.2 in equation 1 gives V = ( 3 a 3 ( 1 - - sc ° s 0) ) + as 3 ' ~i=\~ cnsin0 ° s 00~ )
(2)
The angle between the tangent to a drop and the solid surface at the 3-phase solid/liquid/air meeting point was calculated from the equation ~a 3
V = ~ - (3 t g 0 / 2 + ( t g 0 / 2 ) 3)
(3)
which is identical to equation 2, with the aid of a desk computer (ABC-80, Scandiametric Dator AB, Solna, Sweden). Solutions of the equation for different diameters (2 X a) with a constant volume of 10 pl are given in Table 1. With a known drop volume and diameter the expected height if the shape of the drop is that of a truncated sphere may be calculated from equation 1. The actual height of the drops was measured from the side with a standard microscope equipped with an ocular micrometer.
Preparation of cell monolayers Monolayers of human polymorphonuclear leukocytes (PMNL) were
173
TABLE 1 Solutions of equation 3 (Material and Methods) for 10 pl d r o p s w i t h d i a m e t e r s b e t w e e n 3.50 (0 = 8 5 . 5 ) a n d 8.00 m m (0 = 11.3).
3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5,0 5,1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7,9 8,0
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
85.5 82.1 78.8 75.5 72.2 69.0 65.9 62.9 59.9 57.1 54.4 51.8 49.3 46.9 44.6 42.4 40.3 38.4 36.5 34.8 33.1 31.5 30.0 28.7 27.3 26.1 24.9 23.8 22.7 21.7 20.8 19.9 19.0 18.2 17.5 16.8 16.1 15.4 14.8 14.3 13.7 13.1 12.7 12.2 11.8 11.3
85.2 81.8 78.4 75.1 71.9 68.7 65.6 62.6 59.7 56.8 54.1 51.5 49.0 46.6 44.4 42.2 40.1 38.2 36.4 34.6 33.0 31.4 30.0 28.5 27.2 26.1 24.8 23.7 22.6 21.6 20.7 19.8 19.0 18.2 17.4 16.7 16.0 15.4 14.8 14,2 13.6 13.1 12.6 12.2 11.7
84.8 81.5 78.1 74.8 71.6 68.4 65.3 62.3 59.4 56.6 53.8 51.3 48.8 46.4 44.1 42.0 40.0 38.0 36.2 34.4 32.8 31.2 29.8 28.4 27.1 25.8 24.7 23.6 22.5 21.5 20.6 19.7 18.9 18.1 17.3 16.6 16.0 15.3 14.7 14.1 13.6 13.1 12.6 12.1 11,7
84.5 81.1 77.8 74.5 71.2 68.1 65.0 62.0 59.1 56.3 53.6 51.0 48.5 46.2 43.9 41.8 39.8 37.9 36.0 34.3 32.6 31.1 29.6 28.3 27.1 25.7 24.6 23.5 22.4 21.4 20.5 19.6 18.8 18.0 17.3 16.6 15.9 15.3 14.7 14.1 13.5 13,0 12,5 12.1 11.6
84.2 80.8 77.4 74.2 70.9 67.8 64.7 61.7 58.8 56.0 53.3 50.8 48.3 45.9 43.7 41.6 39.6 37.6 35.8 34.1 32.5 31.0 29.5 28.1 26.8 25.6 24.4 23.4 22.3 21.3 20.4 19.5 18.7 17.9 17.2 16.5 15,8 15,2 14,6 14,0 13.5 13.0 12.5 12.0 11.6
83.8 80.4 77.1 73.8 70.6 67.4 64.4 61.4 58.5 55.7 53.1 50.5 48.0 45.7 43.5 41.4 39.4 37.5 35.6 34.0 32.3 30.8 29.4 28.0 26.7 25.5 24.3 23.4 22.2 21.2 20.3 19.4 18.6 17.9 17.1 16,4 15,8 15.1 14.5 14.0 13.4 12.9 12.4 12.0 11.5
83.5 80.1 76.8 73.5 70.3 67.1 64.1 61.1 58.2 55.5 52.8 50.2 47.8 45.5 43.3 41.6 39.2 37.3 35.5 33.8 32.2 30.6 29.2 27.9 26.6 25.4 24.2 23.1 22.1 21.2 20.2 19.4 18.6 17,8 17.1 16.4 15.7 15.1 14.5 13.9 13.4 12.9 12.4 11.9 11.5
83.1 79.8 76.5 73.2 70.0 66.8 63.8 60.8 57.9 55.2 52.5 50.0 47.6 45.3 43.0 41.0 39.0 37.1 35.3 33.6 32.0 30.5 29.1 27.7 26.5 25.2 24.1 23.0 22.0 21.1 20,1 19,3 18.5 1~]~7 17.0 16.3 15.6 15.0 14.4 13.9 1.3.3 12.8 12.3 11.9 11.4
82.8 79.4 76.1 72.9 69.6 66.5 63.5 60.5 57.7 54.9 52.3 49.7 47.3 45.0 42.8 40.8 38.8 36.9 35.1 33.4 31.9 30.4 29.0 27.6 26.3 25.1 24,0 22,9 21.9 21,0 20.1 19.2 18.4 17.6 16.9 16.2 15.6 15.0 14.4 13.8 13.3 12.8 12.3 11.8 11.4
82.5 79.1 75.8 72.5 69.3 66.2 63.2 60.2 57.4 54.6 52.0 49.5 47.1 44.8 42.6 40.5 38.6 36.7 35.0 33.3 31.7 30.2 28.8 27.5 26.2 25,0 23,9 22.8 21.8 20.9 20.0 19.1 18.3 17.6 16.8 16.2 15.5 14.9 14.3 13.8 13.2 12.7 12.3 11.8 11.4
174
a
ff
ill
.............. Iil l ......
b
surface
Fig. 1. a: sessile drops on a hydrophobic (left) and a hydrophilic surface (right) photographed from the side. b: a geometrical description of the drop as part of a sphere.
prepared by placing a few drops o f hum a n blood on microscope slides. After incubation f o r 15 min at 37°C in a moist chamber, the clots were washed away with saline, leaving the PMNL adhering to the glass. The monolayers were air dried at r o o m t e m p e r a t u r e for 1 h before the c o n t a c t angle measurements were made. Monolayers o f baker's yeast were prepared by placing 0.1 ml volumes of a cell suspension (2 X 107 yeast cells/ml) on microscope slides. The cells were spread like a blood smear, and dried at r o o m t e m p e r a t u r e as described for PMNL {2--3 X 103 yeast cells/mm2).
Opsonization of yeast cells FITC-conjugated yeast particles (Saccharomyces cerevisiae) were prepared as described b y Hed (1977). The yeast cells were incubated at 37°C for 30 min with 20% normal hum a n serum (v/v in Krebs-Ringer phosphate buffer (KRG)). After incubation the particles were washed 3 times in KRG (phagocytosis ex p er ime nt ) or physiological saline (cont act angle measurement) and adjusted to the desired concentration.
Phagocytosis system To the m o n o l a y e r of PMNL, 0.1 ml of yeast suspension (2.5 X 106/ml) was added. After 15 min incubation at 37°C, the yeast suspension was
175 p o u r e d o f f , a n d t h e slides w a s h e d in KI%G. T h e slides w e r e t h e n e x a m i n e d under the microscope, and phagocytosis determined by the fluorescence e x t i n c t i o n m e t h o d d e s c r i b e d b y H e d (1977). RESULTS
Validity of the approximation Volume of the drops. A liquid d r o p o f a n y size, resting o n a solid surface, has a d e f i n i t e c o n t a c t angle (0), i.e., t h e angle b e t w e e n t h e t a n g e n t o f t h e d r o p a n d t h e solid surface at t h e place o f c o n t a c t b e t w e e n liquid, solid a n d gas is c o n s t a n t . T o a l l o w 0 to b e c a l c u l a t e d f r o m t h e value o f the d i a m eter of the drop, the drop must have a known volume. The volume obtained w i t h t h e m i c r o p i p e t t e u s e d was m e a s u r e d b y weighing t h e d r o p s , a n d was f o u n d t o be 9 . 9 8 pl + 0 . 0 6 (S.D.). Measured and expected drop height. For the calculation of 0 from the d r o p d i a m e t e r t h e s h a p e o f t h e d r o p is i m p o r t a n t . T h e d r o p s h a p e m u s t b e t h a t o f a t r u n c a t e d sphere. T h e h e i g h t o f a d r o p , w i t h k n o w n v o l u m e and d i a m e t e r , a n d t h e s h a p e o f a t r u n c a t e d sphere, m a y be c a l c u l a t e d a c c o r d i n g t o e q u a t i o n 1 (Material and M e t h o d s ) . T h e h e i g h t o f d r o p s w i t h k n o w n v o l u m e a n d d i a m e t e r s w a s m e a s u r e d . T h e values f o u n d w e r e in g o o d agreem e n t w i t h t h e h e i g h t e x p e c t e d if t h e s h a p e o f t h e d r o p s was t h a t o f a trunc a t e d s p h e r e (Table 2). Contact angles with different drop volumes. The diameters of drops of increasing v o l u m e o n a tissue c u l t u r e Petri dish w e r e m e a s u r e d , a n d t h e cont a c t angles c a l c u l a t e d . T h e c a l c u l a t e d c o n t a c t angle did n o t c h a n g e signifi c a n t l y u p to a v o l u m e o f 50 pl ( d i a m e t e r 7 . 5 8 m m ) , b u t w i t h larger v o l u m e s t h a n 50 pl t h e s h a p e o f t h e d r o p c h a n g e d f r o m a t r u n c a t e d s p h e r e to a t r u n c a t e d elipse, resulting in u n d e r e s t i m a t i o n o f t h e c o n t a c t angle (Fig. 2). If t h e
TABLE 2 Comparison between the expected drop height, if the shape of the drops is that of a truncated sphere, and the measured drop height (h in Fig. lb'). Surface
Glass b Glass c
Diameter (ram)
7.70 5.55
Contact angle (o)
12.7 32.3
Height Calculated a (ram)
Measured
0.43 1.20
0.45 +- 0.02 d 1.20 + 0.02
a Expected height calculated from the equation V = •/6 " h (3a 2 + h 2) where the volume (V) and the diameter (2a) are known. b Acid washed microscopic slides (76 m m x 26 mm Menzel-Gl~iser, F.R.G.). c Glass cover slips (24 mm X 32 ram, Chance, U.K.). d Mean + S.D.
176
I I
57
I
56 55 54 53
" lb
2'0 3'0 4'0
5'0' 6'0 zb
4.42 5.60 6.40 7.04 7.58 8.19 8.71
Fig. 2. C a l c u l a t i o n o f t h e c o n t a c t angle o n a plastic surface (tissue-culture dish, Flow L a b o r a t o r i e s ) for d r o p s o f d i f f e r e n t volumes. Abscissa: d r o p v o l u m e ( 1 0 - - 7 0 p l ) a n d t h e d i a m e t e r s m e a s u r e d ( 4 . 4 2 - - 8 . 7 1 r a m ) ; o r d i n a t e : calculated c o n t a c t angle (0).
shape approximation can be made for 10 pl drops no differences in calculated 0 should be observed when a smaller drop is used on the same surface. Both on a hydrophilic surface (low 0) and on a hydrophobic surface (high 0) the differences between the contact angles calculated by measuring the diameter of 5 or 10 pl drops were very small (Table 3).
Contact angles o[ cell monolayers The average contact angle of monolayers of human PMNL, prepared by clotting human blood on microscopic slides, was found to be 17.8 ° (Table 4). Untreated yeast particles were found to be more hydrophilic than the phagocytic cells (lower 0 ), while yeast cells opsonized with normal human serum was found to be more hydrophobic than the phagocytic cells (Table 4).
Phagocy tosis The degree of phagocytosis of the different yeast particles used correlated with their respective contact angle. The non-opsonized yeast cells were more hydrophilic and poorly attached to and ingested by the phagocytes. The
TABLE 3 C o n t a c t angles c a l c u l a t e d b y m e a s u r e m e n t o f t h e d i a m e t e r d r o p s w i t h d i f f e r e n t volumes.
Volume of the drop C a l c u l a t e d c o n t a c t angle
H y d r o p h i l i c surface a
H y d r o p h o b i c surface b
5/ll 17.2 -+ 0.7 c
5 pl 89.7 + 0.8
10/21 17.7 + 0.8
a M i c r o s c o p i c slides (76 m m × 26 m m , Menzel-Gl//ser, F.R.G.). b P o l y s t y r o l Petri dishes (90 m m , A / S NUNC, Roskilde, D e n m a r k ) . c M e a n + S.E.M. of 10 e x p e r i m e n t s .
10 pl 90.2 -+ 0.9
177 TABLE 4 Phagocytosis and contact angle measurements on PMNL and yeast cells. Contact angle a
PMNL Non-opsonized yeast Serum-opsonized yeast
17.8 _+0.9 13.3 ± 1.0 26.4 _+1.1
Interaction with PMNL Association b
Ingestion c
-15 ± 3 77 ± 3
-8 ±1 69 ± 4
a Mean + S.E.M. of 10 experiments. b % PMNL with cell associated yeast particles. c % PMNL with intracellularly localized yeast particles.
serum o p s o n i z e d y e a s t cells were m o r e h y d r o p h o b i c and easily a t t a c h e d to and ingested b y the P M N L (Table 4). DISCUSSION Liability to h y d r o p h o b i c i n t e r a c t i o n ( T a n f o r d , 1 9 7 3 ) has b e e n claimed t o b e o f i m p o r t a n c e for h o s t parasite i n t e r a c t i o n (Van Oss et al., 1 9 7 5 ; StjernstrSm et al., 1 9 7 7 ; Magnusson et al., 1 9 7 9 ) and f o r the virulence o f bacteria (Van Oss and Gillman, 1 9 7 2 ; Tagesson and Stendahl, 1973). A n u m b e r o f m e t h o d s have b e e n used to assay h y d r o p h o b i c effects. T o evaluate differences in c o m p a r i s o n to water, 2-phase systems having a high c o n t e n t o f water, such as d e x t r a n - p o l y e t h y l e n e glycol (PEG) systems, can be used (Albertsson, 1971). T o p r o m o t e h y d r o p h o b i c i n t e r a c t i o n , h y d r o p h o b i c PEG, such as P E G - p a l m i t a t e (P-PEG) c o u l d be a d d e d to t h e s y s t e m (Johansson, 1 9 7 6 ) . A n o t h e r m e t h o d , similar to a q u e o u s biphasic p a r t i t i o n i n g with P-PEG in P E G - d e x t r a n systems, is h y d r o p h o b i c i n t e r a c t i o n c h r o m a t o g r a p h y on, for instance, octyl- or p h e n y l - S e p h a r o s e ( R o s e n g r e n et al., 1 9 7 5 ) . Water has a high surface t e n s i o n whereas m o s t h y d r o p h o b i c substances and surfaces have low values. T h e r e f o r e , c o n d e n s a t i o n t e c h n i q u e s (Elwing et al., 1 9 7 7 ) and c o n t a c t angles, i.e., the angles b e t w e e n solid surfaces and liquid d r o p s (Zisman, 1964), can be used to q u a n t i t a t e the average surface t e n s i o n o f biological material (Van Oss et al., 1975). When i n t e r a c t i o n s b e t w e e n large particles, such as p h a g o c y t e s and ingestible material, are at issue, the react i o n b e t w e e n t h e m can be viewed e i t h e r as an overall surface p h e n o m e n o n or as a patch-wise r e a c t i o n b e t w e e n h y d r o p h o b i c surface entities w h i c h are f o r c e d into an a q u e o u s milieu b y d o m i n a n t h y d r o p h i l i c groups. This is an i m p o r t a n t distinction, since d i f f e r e n t m e t h o d s used for p r o b i n g surface h y d r o p h o b i c i t y m a y reflect either the local a f f i n i t y f o r t h e p r o b e or the average surface t e n s i o n (Magnusson, 1 9 8 0 ) . C o n t a c t angles reflecting the average surface t e n s i o n are usually m e a s u r e d b y l o o k i n g at liquid d r o p s f r o m the side t h r o u g h a t e l e s c o p e with crossed
178 hairs attached to a goniometer (Van Oss et al., 1975). The reason for this is that liquid drops on a solid surface have different shapes, depending on the size of the drops, but the change in shape does not affect the contact angle at the 3-phase solid/liquid/air meeting point (Adamson and Ling, 1964). The shape of a very small drop is that of a truncated sphere. For larger sizes, the shape changes through a truncated elipse to a shape with a flat middle for large drops. Assuming that the shape of small drops (10 pl) when put on a flat surface is a truncated sphere, it is possible to calculate the contact angle by measurement of the diameter. A prerequisite for the calculations is that the volume of the drop is known, since deviations in drop volume will lead to miscalculations of the contact angle. The results with volume measurement of drops with the micropipette used indicate that the miscalculation of the contact angle due to volume variations is very small. If the volume of a drop on a flat surface with a contact angle of 20.6 ° is 9.9 pl instead of the expected 10 pl, the calculated contact angle will be 20.8 °. To be able to calculate 0 from the value of the drop diameter, the shape of the drop has to be that of a truncated sphere. If the shape of the drop was a truncated elipse, this would lead to underestimation of the contact angle. The experiments with drops of increasing size show very small changes in the contact angle up to a drop diameter of about 7.58 mm (Fig. 2). For a drop with large volume (larger diameter) the contact angle is underestimated. For a 10 pl drop, a diameter of 7.58 mm corresponds to a contact angle of 13.3 °. A drop on a flat surface will decrease in volume owing to evaporation at a rate depending on the air humidity. We have found that the volume may decrease by 15% in a 10 min period w i t h o u t any measurable change in drop diameter. This indicates that the angle calculated through diameter measurements is the advancing contact angle (Van Oss et al., 1975). If the shape of 10 pl drops used to calculate the contact angle is that of a truncated sphere, the shape of smaller drops would be the same, and the calculated contact angle would be the same if 5 t~l drops were used. Furthermore, the expected height of the drops calculated from equation 2 (Material and Methods) with a known drop diameter and volume should be in agreemerit with the measured drop height. Only very small differences in contact angles, calculated from the diameter of 5 and 10 pl drops, were found (Table 3) and calculated values of the height of the drops were in good agreement with the measured values (Table 2). It has been shown that a correlation exists between the hydrophobicity or tendency to hydrophobic interaction of particles and the degree to which they become phagocytized (Van Oss et al., 1975; Stjernstrhm et al., 1977; Magnusson et al., 1979). Monolayers of human phagocytes (PMNL) have been shown to give rise to a contact angle with saline of 17.5--18.5 ° (Van Oss et al., 1975). Particles with surfaces more hydrophobic than the phagocytic cells (higher 0) are phagocytized while particles with surfaces more hydrophilic than the phagocytes (lower 0) resist phagocytosis. It has also been shown that opsonization by immunoglobulins and/or complement
179 results in an increased h y d r o p h o b i c i t y or t e n d e n c y to h y d r o p h o b i c intera c t i o n , so t h a t n o r m a l l y h y d r o p h i l i c m i c r o o r g a n i s m s m a y b e c o m e e n g u l f e d b y p h a g o c y t e s (Van Oss et al., 1 9 7 5 ; E d e b o et al., 1980). T h e results r e p o r t e d h e r e w h e r e we s h o w t h a t t h e d e g r e e o f p h a g o c y t o s i s o f d i f f e r e n t y e a s t particles c o r r e l a t e s w i t h t h e i r r e s p e c t i v e c o n t a c t angles, are in agreem e n t w i t h t h e results m e n t i o n e d above. F u r t h e r m o r e t h e c o n t a c t angle obt a i n e d o n m o n o l a y e r s o f h u m a n P M N L is in g o o d a g r e e m e n t w i t h t h e value given b y V a n Oss et al. ( 1 9 7 5 ) ( T a b l e 4). T h e results in this r e p o r t s h o w t h a t t h e c o n t a c t angle b e t w e e n a sessile d r o p o f saline a n d a flat l a y e r o f m i c r o o r g a n i s m s or o t h e r cells can b e calc u l a t e d f r o m values o f t h e d r o p d i a m e t e r p r o v i d e d t h e shape o f t h e d r o p s a p p r o x i m a t e s to t h a t o f a t r u n c a t e d sphere. T h e results are r e p r o d u c i b l e and c o n t a c t angles can be e s t i m a t e d w i t h a s t a n d a r d e r r o r o f t h e m e a n o f a r o u n d 1 °. T h e o n l y e q u i p m e n t r e q u i r e d is an o r d i n a r y low m a g n i f y i n g m i c r o s c o p e w i t h an o c u l a r m i c r o m e t e r . ACKNOWLEDGEMENT We w o u d like to t h a n k Dr. Karl-Eric M a g n u s s o n f o r valuable discussions. REFERENCES Adamson, A.W. and I. Ling, 1964, Adv. Chem. Ser. 43, 57. Albertsson, P.-•., 1971, Partition of Cell Particles and Macromolecules, 2nd edn. (Almqvist and Wiksell, Uppsala and Wiley, New York). Edebo, L., E. Kihlstr6m, K.-E. Magnusson and O. Stendahl, 1980, Cell Adhesion and Motility, eds. A.S.G. Curtis and J.D. Pitts (Cambridge University Press, Cambridge) p. 65. Elwing, H., L.-A. Nilsson and O. Ouehterlony, 1977, J. Immunol. Methods 17,131. Hed, J., 1977, FEMS Lett. 1,357. Johansson, G., 1976, Biochim. Biophys. Acta 451,517. Magnusson, K.-E., 1980, Scand. J. Infect. Dis. In press. Magnusson, K.-E., O. Stendahl, C. Tagesson, L. Edebo and G. Johansson, 1977, Acta Pathol. Microbiol. Scand. Section B 85,212. Magnusson, K.-E., O. Stendahl, I. Stjernstr6m and L. Edebo, 1979, Immunology 36,439. Rosengren, J., S. P~hlman, M. Glad and S. Hjert6n, 1975, Biochim. Biophys. Acta 413, 51. Standard Mathematical Tables, 20th edn., 1972, Editor-in-chief S.M. Selby (The Chemical Rubber Co., Cleveland, OH). Stjernstr6m, I., K.-E. Magnusson, O. Stendahl and C. Tagesson, 1977, Infect. Immun. 18, 261. Tagesson, C. and O. Stendahl, 1973, Acta Pathol. Microbiol. Scand. Section B 81,473. Tanford, C., 1973, The Hydrophobic Effect. Formation of Micelles and Biological Membranes (Wiley, New York). Van Oss, C.J., 1978, Ann. Rev. Microbiol. 32, 19. Van Oss, C.J. and C.F. Gillman, 1972, J. Reticuloendothel. Soc. 12, 497. Van Oss, C.J., C.F. Gillman and A.W. Neuman, 1975, Phagocytic Engulfment and Cell Adhesiveness as Cellular Surface Phenomena (Marcel Dekker, New York). Zisman, W.A., 1964, Adv. Chem. Ser. 43, 1.
171
PHAGOCYTOSIS AND HYDROPHOBICITY: A METHOD OF C A L C U L A T I N G CONTACT A N G L E S BASED ON THE D I A M E T E R OF SESSILE DROPS
C. DAHLGREN and T. SUNQVIST Department of Medical Microbiology, The University of Link6ping, S-581 85 Link6ping, Sweden
(Received 12 May 1980, accepted 1 September 1980)
The correlation between the contact angle and degree of phagocytosis of different yeast particles has been investigated. To facilitate the estimation of the contact angle, we have tested the hypothesis that the shape of a small liquid drop put on a flat surface is that of a truncated sphere. By making this approximation it is possible to calculate the contact angle, i.e. the tangent to the drop in the 3-phase liquid/solid/air meeting point, by measuring the drop diameter. Known volumes of saline were put on different surfaces and the diameters of the drops were measured from above. Calculation of the contact angle with drops of different volumes, and comparison between expected and measured height of 10 pl drops, indicated that the assumption that the shape of a drop is that of a truncated sphere is valid. Monolayers of leukocytes was shown to give rise to a contact angle of 17.9 °. Particles with a lower contact angle than the phagocytic cells resisted phagocytosis, but opsonization of the particles with normal human serum rendered them susceptible to phagocytosis, conferring a higher contact angle than that of the phagocytic cells.
INTRODUCTION The process o f p h a g o c y t o s i s is initiated b y a t t a c h m e n t o f t h e p r e y t o t h e surface o f t h e p h a g o c y t i c cell, f o l l o w e d b y its e n g u l f m e n t and d e g r a d a t i o n . S t u d y o f the p h y s i c o - c h e m i c a l surface p r o p e r t i e s of bacteria in relation to their liability o f p h a g o c y t o s i s has s h o w n t h a t the t e n d e n c y t o h y d r o p h o b i c i n t e r a c t i o n is o f i m p o r t a n c e f o r host-parasite i n t e r a c t i o n (Van Oss et al., 1 9 7 5 ; S t j e r n s t r S m et al., 1 9 7 7 ; M a g n u s s o n et al., 1 9 7 9 ) and for virulence o f t h e b a c t e r i a (Van Oss and Gillman, 1 9 7 2 ; Tagesson and Stendahl, 1973}. D i f f e r e n t m e t h o d s o f e s t i m a t i n g h y d r o p h o b i c i t y have b e e n used t o correlate this w i t h the virulence o f m i c r o o r g a n i s m s (Magnusson et al., 1 9 7 7 ; S t j e r n s t r S m et al., 1 9 7 7 ; V a n Oss, 1 9 7 8 ) . T h e c o n t a c t angle, i.e., the angle f o r m e d b e t w e e n a sessile d r o p o f saline and a flat surface c a r r y i n g a m o n o l a y e r o f cells, has been used as a m e a s u r e o f surface h y d r o p h o b i c i t y (Van Oss and Gillman., 1 9 7 2 ; V a n Oss et al., 1 9 7 5 ; V a n Oss, 1 9 7 8 ) . C o n t a c t angles are usually m e a s u r e d w i t h t h e aid o f a t e l e s c o p e w i t h Crossed hairs
172
attached to a goniometer. In order to simplify the measurement of contact angles, we have tested the approximation that the shape of a drop of saline on a flat surface is that of a truncated sphere, and found that the diameter of a drop of saline on a flat surface can be used to calculate the contact angle. MATERIAL
AND METHODS
Contact angle measurements Known volumes (V) of saline were placed on different surfaces and the diameters of the drops were measured from above with a microscope equipped with an ocular micrometer (Zeiss stereomicroscope with a zoom system). It was hypothesized that the shape of the drop may be approximated to that of a truncated sphere. The volume (V) of a truncated sphere (Standard Mathematical Tables) is
=g~ h ( 3 a 2 + h 2)
V
(1)
where the symbols correspond to those in Fig. 1. The height may be described as
h
a
a
sin 0
tg 0
-
(1.2)
and a substitution of equation 1.2 in equation 1 gives V = ( 3 a 3 ( 1 - - sc ° s 0) ) + as 3 ' ~i=\~ cnsin0 ° s 00~ )
(2)
The angle between the tangent to a drop and the solid surface at the 3-phase solid/liquid/air meeting point was calculated from the equation ~a 3
V = ~ - (3 t g 0 / 2 + ( t g 0 / 2 ) 3)
(3)
which is identical to equation 2, with the aid of a desk computer (ABC-80, Scandiametric Dator AB, Solna, Sweden). Solutions of the equation for different diameters (2 X a) with a constant volume of 10 pl are given in Table 1. With a known drop volume and diameter the expected height if the shape of the drop is that of a truncated sphere may be calculated from equation 1. The actual height of the drops was measured from the side with a standard microscope equipped with an ocular micrometer.
Preparation of cell monolayers Monolayers of human polymorphonuclear leukocytes (PMNL) were
173
TABLE 1 Solutions of equation 3 (Material and Methods) for 10 pl d r o p s w i t h d i a m e t e r s b e t w e e n 3.50 (0 = 8 5 . 5 ) a n d 8.00 m m (0 = 11.3).
3.5 3.6 3.7 3.8 3.9 4.0 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 5,0 5,1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7,9 8,0
0.00
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
85.5 82.1 78.8 75.5 72.2 69.0 65.9 62.9 59.9 57.1 54.4 51.8 49.3 46.9 44.6 42.4 40.3 38.4 36.5 34.8 33.1 31.5 30.0 28.7 27.3 26.1 24.9 23.8 22.7 21.7 20.8 19.9 19.0 18.2 17.5 16.8 16.1 15.4 14.8 14.3 13.7 13.1 12.7 12.2 11.8 11.3
85.2 81.8 78.4 75.1 71.9 68.7 65.6 62.6 59.7 56.8 54.1 51.5 49.0 46.6 44.4 42.2 40.1 38.2 36.4 34.6 33.0 31.4 30.0 28.5 27.2 26.1 24.8 23.7 22.6 21.6 20.7 19.8 19.0 18.2 17.4 16.7 16.0 15.4 14.8 14,2 13.6 13.1 12.6 12.2 11.7
84.8 81.5 78.1 74.8 71.6 68.4 65.3 62.3 59.4 56.6 53.8 51.3 48.8 46.4 44.1 42.0 40.0 38.0 36.2 34.4 32.8 31.2 29.8 28.4 27.1 25.8 24.7 23.6 22.5 21.5 20.6 19.7 18.9 18.1 17.3 16.6 16.0 15.3 14.7 14.1 13.6 13.1 12.6 12.1 11,7
84.5 81.1 77.8 74.5 71.2 68.1 65.0 62.0 59.1 56.3 53.6 51.0 48.5 46.2 43.9 41.8 39.8 37.9 36.0 34.3 32.6 31.1 29.6 28.3 27.1 25.7 24.6 23.5 22.4 21.4 20.5 19.6 18.8 18.0 17.3 16.6 15.9 15.3 14.7 14.1 13.5 13,0 12,5 12.1 11.6
84.2 80.8 77.4 74.2 70.9 67.8 64.7 61.7 58.8 56.0 53.3 50.8 48.3 45.9 43.7 41.6 39.6 37.6 35.8 34.1 32.5 31.0 29.5 28.1 26.8 25.6 24.4 23.4 22.3 21.3 20.4 19.5 18.7 17.9 17.2 16.5 15,8 15,2 14,6 14,0 13.5 13.0 12.5 12.0 11.6
83.8 80.4 77.1 73.8 70.6 67.4 64.4 61.4 58.5 55.7 53.1 50.5 48.0 45.7 43.5 41.4 39.4 37.5 35.6 34.0 32.3 30.8 29.4 28.0 26.7 25.5 24.3 23.4 22.2 21.2 20.3 19.4 18.6 17.9 17.1 16,4 15,8 15.1 14.5 14.0 13.4 12.9 12.4 12.0 11.5
83.5 80.1 76.8 73.5 70.3 67.1 64.1 61.1 58.2 55.5 52.8 50.2 47.8 45.5 43.3 41.6 39.2 37.3 35.5 33.8 32.2 30.6 29.2 27.9 26.6 25.4 24.2 23.1 22.1 21.2 20.2 19.4 18.6 17,8 17.1 16.4 15.7 15.1 14.5 13.9 13.4 12.9 12.4 11.9 11.5
83.1 79.8 76.5 73.2 70.0 66.8 63.8 60.8 57.9 55.2 52.5 50.0 47.6 45.3 43.0 41.0 39.0 37.1 35.3 33.6 32.0 30.5 29.1 27.7 26.5 25.2 24.1 23.0 22.0 21.1 20,1 19,3 18.5 1~]~7 17.0 16.3 15.6 15.0 14.4 13.9 1.3.3 12.8 12.3 11.9 11.4
82.8 79.4 76.1 72.9 69.6 66.5 63.5 60.5 57.7 54.9 52.3 49.7 47.3 45.0 42.8 40.8 38.8 36.9 35.1 33.4 31.9 30.4 29.0 27.6 26.3 25.1 24,0 22,9 21.9 21,0 20.1 19.2 18.4 17.6 16.9 16.2 15.6 15.0 14.4 13.8 13.3 12.8 12.3 11.8 11.4
82.5 79.1 75.8 72.5 69.3 66.2 63.2 60.2 57.4 54.6 52.0 49.5 47.1 44.8 42.6 40.5 38.6 36.7 35.0 33.3 31.7 30.2 28.8 27.5 26.2 25,0 23,9 22.8 21.8 20.9 20.0 19.1 18.3 17.6 16.8 16.2 15.5 14.9 14.3 13.8 13.2 12.7 12.3 11.8 11.4
174
a
ff
ill
.............. Iil l ......
b
surface
Fig. 1. a: sessile drops on a hydrophobic (left) and a hydrophilic surface (right) photographed from the side. b: a geometrical description of the drop as part of a sphere.
prepared by placing a few drops o f hum a n blood on microscope slides. After incubation f o r 15 min at 37°C in a moist chamber, the clots were washed away with saline, leaving the PMNL adhering to the glass. The monolayers were air dried at r o o m t e m p e r a t u r e for 1 h before the c o n t a c t angle measurements were made. Monolayers o f baker's yeast were prepared by placing 0.1 ml volumes of a cell suspension (2 X 107 yeast cells/ml) on microscope slides. The cells were spread like a blood smear, and dried at r o o m t e m p e r a t u r e as described for PMNL {2--3 X 103 yeast cells/mm2).
Opsonization of yeast cells FITC-conjugated yeast particles (Saccharomyces cerevisiae) were prepared as described b y Hed (1977). The yeast cells were incubated at 37°C for 30 min with 20% normal hum a n serum (v/v in Krebs-Ringer phosphate buffer (KRG)). After incubation the particles were washed 3 times in KRG (phagocytosis ex p er ime nt ) or physiological saline (cont act angle measurement) and adjusted to the desired concentration.
Phagocytosis system To the m o n o l a y e r of PMNL, 0.1 ml of yeast suspension (2.5 X 106/ml) was added. After 15 min incubation at 37°C, the yeast suspension was
175 p o u r e d o f f , a n d t h e slides w a s h e d in KI%G. T h e slides w e r e t h e n e x a m i n e d under the microscope, and phagocytosis determined by the fluorescence e x t i n c t i o n m e t h o d d e s c r i b e d b y H e d (1977). RESULTS
Validity of the approximation Volume of the drops. A liquid d r o p o f a n y size, resting o n a solid surface, has a d e f i n i t e c o n t a c t angle (0), i.e., t h e angle b e t w e e n t h e t a n g e n t o f t h e d r o p a n d t h e solid surface at t h e place o f c o n t a c t b e t w e e n liquid, solid a n d gas is c o n s t a n t . T o a l l o w 0 to b e c a l c u l a t e d f r o m t h e value o f the d i a m eter of the drop, the drop must have a known volume. The volume obtained w i t h t h e m i c r o p i p e t t e u s e d was m e a s u r e d b y weighing t h e d r o p s , a n d was f o u n d t o be 9 . 9 8 pl + 0 . 0 6 (S.D.). Measured and expected drop height. For the calculation of 0 from the d r o p d i a m e t e r t h e s h a p e o f t h e d r o p is i m p o r t a n t . T h e d r o p s h a p e m u s t b e t h a t o f a t r u n c a t e d sphere. T h e h e i g h t o f a d r o p , w i t h k n o w n v o l u m e and d i a m e t e r , a n d t h e s h a p e o f a t r u n c a t e d sphere, m a y be c a l c u l a t e d a c c o r d i n g t o e q u a t i o n 1 (Material and M e t h o d s ) . T h e h e i g h t o f d r o p s w i t h k n o w n v o l u m e a n d d i a m e t e r s w a s m e a s u r e d . T h e values f o u n d w e r e in g o o d agreem e n t w i t h t h e h e i g h t e x p e c t e d if t h e s h a p e o f t h e d r o p s was t h a t o f a trunc a t e d s p h e r e (Table 2). Contact angles with different drop volumes. The diameters of drops of increasing v o l u m e o n a tissue c u l t u r e Petri dish w e r e m e a s u r e d , a n d t h e cont a c t angles c a l c u l a t e d . T h e c a l c u l a t e d c o n t a c t angle did n o t c h a n g e signifi c a n t l y u p to a v o l u m e o f 50 pl ( d i a m e t e r 7 . 5 8 m m ) , b u t w i t h larger v o l u m e s t h a n 50 pl t h e s h a p e o f t h e d r o p c h a n g e d f r o m a t r u n c a t e d s p h e r e to a t r u n c a t e d elipse, resulting in u n d e r e s t i m a t i o n o f t h e c o n t a c t angle (Fig. 2). If t h e
TABLE 2 Comparison between the expected drop height, if the shape of the drops is that of a truncated sphere, and the measured drop height (h in Fig. lb'). Surface
Glass b Glass c
Diameter (ram)
7.70 5.55
Contact angle (o)
12.7 32.3
Height Calculated a (ram)
Measured
0.43 1.20
0.45 +- 0.02 d 1.20 + 0.02
a Expected height calculated from the equation V = •/6 " h (3a 2 + h 2) where the volume (V) and the diameter (2a) are known. b Acid washed microscopic slides (76 m m x 26 mm Menzel-Gl~iser, F.R.G.). c Glass cover slips (24 mm X 32 ram, Chance, U.K.). d Mean + S.D.
176
I I
57
I
56 55 54 53
" lb
2'0 3'0 4'0
5'0' 6'0 zb
4.42 5.60 6.40 7.04 7.58 8.19 8.71
Fig. 2. C a l c u l a t i o n o f t h e c o n t a c t angle o n a plastic surface (tissue-culture dish, Flow L a b o r a t o r i e s ) for d r o p s o f d i f f e r e n t volumes. Abscissa: d r o p v o l u m e ( 1 0 - - 7 0 p l ) a n d t h e d i a m e t e r s m e a s u r e d ( 4 . 4 2 - - 8 . 7 1 r a m ) ; o r d i n a t e : calculated c o n t a c t angle (0).
shape approximation can be made for 10 pl drops no differences in calculated 0 should be observed when a smaller drop is used on the same surface. Both on a hydrophilic surface (low 0) and on a hydrophobic surface (high 0) the differences between the contact angles calculated by measuring the diameter of 5 or 10 pl drops were very small (Table 3).
Contact angles o[ cell monolayers The average contact angle of monolayers of human PMNL, prepared by clotting human blood on microscopic slides, was found to be 17.8 ° (Table 4). Untreated yeast particles were found to be more hydrophilic than the phagocytic cells (lower 0 ), while yeast cells opsonized with normal human serum was found to be more hydrophobic than the phagocytic cells (Table 4).
Phagocy tosis The degree of phagocytosis of the different yeast particles used correlated with their respective contact angle. The non-opsonized yeast cells were more hydrophilic and poorly attached to and ingested by the phagocytes. The
TABLE 3 C o n t a c t angles c a l c u l a t e d b y m e a s u r e m e n t o f t h e d i a m e t e r d r o p s w i t h d i f f e r e n t volumes.
Volume of the drop C a l c u l a t e d c o n t a c t angle
H y d r o p h i l i c surface a
H y d r o p h o b i c surface b
5/ll 17.2 -+ 0.7 c
5 pl 89.7 + 0.8
10/21 17.7 + 0.8
a M i c r o s c o p i c slides (76 m m × 26 m m , Menzel-Gl//ser, F.R.G.). b P o l y s t y r o l Petri dishes (90 m m , A / S NUNC, Roskilde, D e n m a r k ) . c M e a n + S.E.M. of 10 e x p e r i m e n t s .
10 pl 90.2 -+ 0.9
177 TABLE 4 Phagocytosis and contact angle measurements on PMNL and yeast cells. Contact angle a
PMNL Non-opsonized yeast Serum-opsonized yeast
17.8 _+0.9 13.3 ± 1.0 26.4 _+1.1
Interaction with PMNL Association b
Ingestion c
-15 ± 3 77 ± 3
-8 ±1 69 ± 4
a Mean + S.E.M. of 10 experiments. b % PMNL with cell associated yeast particles. c % PMNL with intracellularly localized yeast particles.
serum o p s o n i z e d y e a s t cells were m o r e h y d r o p h o b i c and easily a t t a c h e d to and ingested b y the P M N L (Table 4). DISCUSSION Liability to h y d r o p h o b i c i n t e r a c t i o n ( T a n f o r d , 1 9 7 3 ) has b e e n claimed t o b e o f i m p o r t a n c e for h o s t parasite i n t e r a c t i o n (Van Oss et al., 1 9 7 5 ; StjernstrSm et al., 1 9 7 7 ; Magnusson et al., 1 9 7 9 ) and f o r the virulence o f bacteria (Van Oss and Gillman, 1 9 7 2 ; Tagesson and Stendahl, 1973). A n u m b e r o f m e t h o d s have b e e n used to assay h y d r o p h o b i c effects. T o evaluate differences in c o m p a r i s o n to water, 2-phase systems having a high c o n t e n t o f water, such as d e x t r a n - p o l y e t h y l e n e glycol (PEG) systems, can be used (Albertsson, 1971). T o p r o m o t e h y d r o p h o b i c i n t e r a c t i o n , h y d r o p h o b i c PEG, such as P E G - p a l m i t a t e (P-PEG) c o u l d be a d d e d to t h e s y s t e m (Johansson, 1 9 7 6 ) . A n o t h e r m e t h o d , similar to a q u e o u s biphasic p a r t i t i o n i n g with P-PEG in P E G - d e x t r a n systems, is h y d r o p h o b i c i n t e r a c t i o n c h r o m a t o g r a p h y on, for instance, octyl- or p h e n y l - S e p h a r o s e ( R o s e n g r e n et al., 1 9 7 5 ) . Water has a high surface t e n s i o n whereas m o s t h y d r o p h o b i c substances and surfaces have low values. T h e r e f o r e , c o n d e n s a t i o n t e c h n i q u e s (Elwing et al., 1 9 7 7 ) and c o n t a c t angles, i.e., the angles b e t w e e n solid surfaces and liquid d r o p s (Zisman, 1964), can be used to q u a n t i t a t e the average surface t e n s i o n o f biological material (Van Oss et al., 1975). When i n t e r a c t i o n s b e t w e e n large particles, such as p h a g o c y t e s and ingestible material, are at issue, the react i o n b e t w e e n t h e m can be viewed e i t h e r as an overall surface p h e n o m e n o n or as a patch-wise r e a c t i o n b e t w e e n h y d r o p h o b i c surface entities w h i c h are f o r c e d into an a q u e o u s milieu b y d o m i n a n t h y d r o p h i l i c groups. This is an i m p o r t a n t distinction, since d i f f e r e n t m e t h o d s used for p r o b i n g surface h y d r o p h o b i c i t y m a y reflect either the local a f f i n i t y f o r t h e p r o b e or the average surface t e n s i o n (Magnusson, 1 9 8 0 ) . C o n t a c t angles reflecting the average surface t e n s i o n are usually m e a s u r e d b y l o o k i n g at liquid d r o p s f r o m the side t h r o u g h a t e l e s c o p e with crossed
178 hairs attached to a goniometer (Van Oss et al., 1975). The reason for this is that liquid drops on a solid surface have different shapes, depending on the size of the drops, but the change in shape does not affect the contact angle at the 3-phase solid/liquid/air meeting point (Adamson and Ling, 1964). The shape of a very small drop is that of a truncated sphere. For larger sizes, the shape changes through a truncated elipse to a shape with a flat middle for large drops. Assuming that the shape of small drops (10 pl) when put on a flat surface is a truncated sphere, it is possible to calculate the contact angle by measurement of the diameter. A prerequisite for the calculations is that the volume of the drop is known, since deviations in drop volume will lead to miscalculations of the contact angle. The results with volume measurement of drops with the micropipette used indicate that the miscalculation of the contact angle due to volume variations is very small. If the volume of a drop on a flat surface with a contact angle of 20.6 ° is 9.9 pl instead of the expected 10 pl, the calculated contact angle will be 20.8 °. To be able to calculate 0 from the value of the drop diameter, the shape of the drop has to be that of a truncated sphere. If the shape of the drop was a truncated elipse, this would lead to underestimation of the contact angle. The experiments with drops of increasing size show very small changes in the contact angle up to a drop diameter of about 7.58 mm (Fig. 2). For a drop with large volume (larger diameter) the contact angle is underestimated. For a 10 pl drop, a diameter of 7.58 mm corresponds to a contact angle of 13.3 °. A drop on a flat surface will decrease in volume owing to evaporation at a rate depending on the air humidity. We have found that the volume may decrease by 15% in a 10 min period w i t h o u t any measurable change in drop diameter. This indicates that the angle calculated through diameter measurements is the advancing contact angle (Van Oss et al., 1975). If the shape of 10 pl drops used to calculate the contact angle is that of a truncated sphere, the shape of smaller drops would be the same, and the calculated contact angle would be the same if 5 t~l drops were used. Furthermore, the expected height of the drops calculated from equation 2 (Material and Methods) with a known drop diameter and volume should be in agreemerit with the measured drop height. Only very small differences in contact angles, calculated from the diameter of 5 and 10 pl drops, were found (Table 3) and calculated values of the height of the drops were in good agreement with the measured values (Table 2). It has been shown that a correlation exists between the hydrophobicity or tendency to hydrophobic interaction of particles and the degree to which they become phagocytized (Van Oss et al., 1975; Stjernstrhm et al., 1977; Magnusson et al., 1979). Monolayers of human phagocytes (PMNL) have been shown to give rise to a contact angle with saline of 17.5--18.5 ° (Van Oss et al., 1975). Particles with surfaces more hydrophobic than the phagocytic cells (higher 0) are phagocytized while particles with surfaces more hydrophilic than the phagocytes (lower 0) resist phagocytosis. It has also been shown that opsonization by immunoglobulins and/or complement
179 results in an increased h y d r o p h o b i c i t y or t e n d e n c y to h y d r o p h o b i c intera c t i o n , so t h a t n o r m a l l y h y d r o p h i l i c m i c r o o r g a n i s m s m a y b e c o m e e n g u l f e d b y p h a g o c y t e s (Van Oss et al., 1 9 7 5 ; E d e b o et al., 1980). T h e results r e p o r t e d h e r e w h e r e we s h o w t h a t t h e d e g r e e o f p h a g o c y t o s i s o f d i f f e r e n t y e a s t particles c o r r e l a t e s w i t h t h e i r r e s p e c t i v e c o n t a c t angles, are in agreem e n t w i t h t h e results m e n t i o n e d above. F u r t h e r m o r e t h e c o n t a c t angle obt a i n e d o n m o n o l a y e r s o f h u m a n P M N L is in g o o d a g r e e m e n t w i t h t h e value given b y V a n Oss et al. ( 1 9 7 5 ) ( T a b l e 4). T h e results in this r e p o r t s h o w t h a t t h e c o n t a c t angle b e t w e e n a sessile d r o p o f saline a n d a flat l a y e r o f m i c r o o r g a n i s m s or o t h e r cells can b e calc u l a t e d f r o m values o f t h e d r o p d i a m e t e r p r o v i d e d t h e shape o f t h e d r o p s a p p r o x i m a t e s to t h a t o f a t r u n c a t e d sphere. T h e results are r e p r o d u c i b l e and c o n t a c t angles can be e s t i m a t e d w i t h a s t a n d a r d e r r o r o f t h e m e a n o f a r o u n d 1 °. T h e o n l y e q u i p m e n t r e q u i r e d is an o r d i n a r y low m a g n i f y i n g m i c r o s c o p e w i t h an o c u l a r m i c r o m e t e r . ACKNOWLEDGEMENT We w o u d like to t h a n k Dr. Karl-Eric M a g n u s s o n f o r valuable discussions. REFERENCES Adamson, A.W. and I. Ling, 1964, Adv. Chem. Ser. 43, 57. Albertsson, P.-•., 1971, Partition of Cell Particles and Macromolecules, 2nd edn. (Almqvist and Wiksell, Uppsala and Wiley, New York). Edebo, L., E. Kihlstr6m, K.-E. Magnusson and O. Stendahl, 1980, Cell Adhesion and Motility, eds. A.S.G. Curtis and J.D. Pitts (Cambridge University Press, Cambridge) p. 65. Elwing, H., L.-A. Nilsson and O. Ouehterlony, 1977, J. Immunol. Methods 17,131. Hed, J., 1977, FEMS Lett. 1,357. Johansson, G., 1976, Biochim. Biophys. Acta 451,517. Magnusson, K.-E., 1980, Scand. J. Infect. Dis. In press. Magnusson, K.-E., O. Stendahl, C. Tagesson, L. Edebo and G. Johansson, 1977, Acta Pathol. Microbiol. Scand. Section B 85,212. Magnusson, K.-E., O. Stendahl, I. Stjernstr6m and L. Edebo, 1979, Immunology 36,439. Rosengren, J., S. P~hlman, M. Glad and S. Hjert6n, 1975, Biochim. Biophys. Acta 413, 51. Standard Mathematical Tables, 20th edn., 1972, Editor-in-chief S.M. Selby (The Chemical Rubber Co., Cleveland, OH). Stjernstr6m, I., K.-E. Magnusson, O. Stendahl and C. Tagesson, 1977, Infect. Immun. 18, 261. Tagesson, C. and O. Stendahl, 1973, Acta Pathol. Microbiol. Scand. Section B 81,473. Tanford, C., 1973, The Hydrophobic Effect. Formation of Micelles and Biological Membranes (Wiley, New York). Van Oss, C.J., 1978, Ann. Rev. Microbiol. 32, 19. Van Oss, C.J. and C.F. Gillman, 1972, J. Reticuloendothel. Soc. 12, 497. Van Oss, C.J., C.F. Gillman and A.W. Neuman, 1975, Phagocytic Engulfment and Cell Adhesiveness as Cellular Surface Phenomena (Marcel Dekker, New York). Zisman, W.A., 1964, Adv. Chem. Ser. 43, 1.