CELL BIOPHYSICS 2, 113-126 (1980)

Surface Thermodynamics of Normal and Pathological Human Granulocytes

D . R . A B S O L O M , *a'b C . J . VAN O S S , a g . J . G E N C O , c

D . W . FRANCIS, b AND A . W . N E U M A N N b

aDepartment of Microbiology State University of New York at Buffalo Buffalo, New York 14214

bDepartment of Mechanical Engineering University of Toronto Toronto, Ontario M5S 1A4 Canada

CDepartment of Oral Biology State University of New York at Buffalo Buffalo, New York 14214

Received June 6, 1979; Accepted January 3, 1980

Abstract

Surface tensions of normal and pathological granulocytes were determined by (1) adhesion to solid substrates of different surface tensions while suspended in liquid media of different surface tensions, and by (2) measurement of cell-liquid-vapor contact angles obtained with sessile drops of saline water on cell monolayers. The results obtained by the two different methods were in close conformation with one another. With the cell adhesion method some residual leukocyte adhesion still persists even under conditions where there no longer is a van der Waals attraction between cells and solid substrate. At low ionic strength and by the abolishment of all multivalent cations through the admixture of EDTA, that residual cell adhesion virtually disappears (with normal as well as with pathological granulocytes), indicating that the earlier residual cell adhesion did indeed arise from electrostatic interactions mediated by multivalent cations (probably Ca2+). Comparison of the capacities for engulfment and the surface thermodynamics data of normal and pathological granulocytes obtained in this study leads to the novel

�9 1980 The Humana Press Inc. All rights of any nature whatsoever reserved. 0163~,992/80/0600~) 111 $02.80

113

114 ABSOLOM ET AL.

observation that the phagocytic episode from half to complete engulfment of bacterial particles by granulocytes appears to be the crucial step from the thermodynamic point of view.

Index Entries: Cell adhesicn, of normal and pathological granulocytes; leukocytes, normal and pathological, cell adhesion in; granulocytes, normal and pathological, cell adhesion in; cellular surface tension, of normal and pathological granulocytes; cell surface thermodynamics, of normal and pathological granulocytes; free energy of engulfment, of normal and pathological granulocytes; phagocytic activity, of normal and pathological granulocytes.

I n t r o d u c t i o n

Until recently, there were only two different methods for the determination of surface tensions of living cells.

One is based on the determination of the critical surface tensions ('yc), with the method developed by Fox and Zisman (1, 2) and Zisman (3-5). By this method the cosines of the contact angles (0) found (on cell layers) with drops of liquids of various different surface tensions are plotted versus the surface tension of each liquid. At the point of extrapolation to cos 0 = 1, one finds the critical surface tension %. The considerable drawback of this method, when used with living cells, is that, because most living cells are rather hydrophilic, only liquids can be used that are even more hydrophilic than the cells (in order to get a measurable value for 0). This limits the method to the use of water, and water in which the surface tension has been increased by the admixture of considerable amounts of salts [e.g., 0.85%, 8.5%, 17%, 34% NaNO3 (7, 8)], which of course gives rise to fairly extreme osmotic conditions.

The second method uses only contact angles of one liquid (i.e., with living cells, drops of isotonic saline), and allows the derivation of the surface tension of cell surfaces from the data obtained, via the equation of state approach (7, 9). Virtually all the work on the surface properties of normal human peripheral granulocytes has been done by this method; normal human granulocytes were found to have contact angles of 18.0 ~ +0.5 ~ SE (7, 8, 10) corresponding to a surface tension 3/Gv, (G standing for granulocytes and V for vapor) of 69.15 + 0.17 SE ergs/cm 2. (9, 11).

Our third new method differs fundamentally from the first two. Instead of contact angle measurements on living cells, the adhesion of leukocytes to surfaces of various surface tensions, while suspended in liquids of various different surface tensions is determined. By plotting the slopes (of the number of leukocytes adhering per unit surface area versus the surface

*1 erg/cm 2 = 1 milliJoule/m 2.

SURFACE THERMODYNAMICS OF GRANULOCYTES 1 15

tensions of the materials they were adhering to) versus the surface tensions of the liquids the leukocytes were suspended in, the intercept at zero slope corresponds to the surface tension of the liquid at which precisely the same number of leukocytes per unit of surface area would adhere to all materials, regardless of the different surface tensions of these materials. The surface tension of that hypothetical liquid "yLV, (L standing for liquid and V for vapor) then also is the surface tension 3/~v of the granulocytes (11). Granulocytes are the type of leukocytes of which the surface tension has been studied most (7, 8, 10-13). In this manner, it was found that the surface tension of normal human peripheral granulocytes "yGv = 69.0 ergs/cm z) (11), which conforms quite well to "y~v = 69.15 ergs / cm 2 found by means of the saline water drop contact angle method (see above).

It would be useful to make a further comparison between the two methods by doing similar determinations with closely related leukocytes that nevertheless have a significantly different surface tension from the ones studied earlier. Such leukocytes exist, in the form of the peripheral granulocytes of a group of young patients with recurrent localized infections (12), and of patients with idiopathic juvenile peridontal disease (12, 13). In the present work the peripheral granulocytes of patients of the latter class are the object of further studies.

In the earlier study it was also found that under conditions where van der Waals attractions could not be operative, a fairly small but nevertheless positive level of leukocyte adhesion still could be measured (11). There were indications that ionic interactions play a role in that phenomenon. In the present investigation both normal and pathological (13) granulocytes are used in a further attempt to elucidate the nature of that paradoxical residual leukocyte adhesion.

Experimental Procedures

Phagocytic ingestion was determined with Staphylococcus aureus, opsonized with 1% human IgG, at a concentration of 2 • l0 s bacteria/mL, ingested by human granulocytes deposited in a monolayer in Mackaness chambers. Staphylococcus aureus (grown at 37~ on Difco tryptose phosphate broth) were harvested by centrifugation, subjected to two successive saline washes, and resuspended in Hanks' balanced salt solution. For opsonization, the bacteria were incubated for 30 min at 37~ in a solution of pooled human IgG (1% Cohn's Fraction II, obtained from Pentex/Miles, Kanakee, I1.) in saline, and then washed once more prior to being subjected to phagocytosis. Phagocytosis in vitro of opsonized bacteria by human granulocytes was studied in Mackaness coverslip chambers (13). Monolayers of peripheral leukocytes from a few drops of finger prick blood

116 ABSOLOM ET AL.

from a single donor were prepared on round glass coverslips (2.2 cm diameter) as described earlier (13). The resulting cell sheets, containing approximately 200-300 cells/mm 2, were placed into these chambers and exposed to 0.8mL of bacterial (2 X 10 s bacteria/mL) suspension at 37 ~ C for 15 min. After the incubation period the chambers were tapped on their sides to dislodge any free particles adhering to the surface of the monolayers. The coverslips were then excised, washed gently in saline, air dried, and stained. Gram's stain was used to detect the phagocytized bacteria. The total number of granulocytes and the total number of cells having engaged in phagocytosis, as well as the number of bacteria ingested per cell, were counted on two traverses on the monolayers taken at right angles to each other through the center of the coverslip. A total of 40 cells was counted. We designated as the phagocytic activity the mean number of bacteria phagocytized per granulocyte and calculated the standard error of the mean (7, 8, 10).

Contact angle determinations on the monolayer of cells were determined using isotonic saline as described previously (8). Briefly the technique involves the measurement of the contact angle made between a sessile drop of liquid and a layer of solid material in air (3-5). This technique was adopted and the contact angles of sessile drops of saline with monolayers of granulocytes, formed as described above, were measured using a telescope with crosshairs, attached to a goniometer (Gaerther Scientific Corp., Chicago, Ill.). The angles of at least 10 sessile drops of physiological saline are measured, and the averages taken; the results are quite reproducible and the contact angles can, with care, be estimated within + < 1 ~ Some deviation from complete flatness is bound to persist and to cause a certain unavoidable degree of error in the contact-angle measurements. However, since the diameter of the saline drop is larger than that of single cells by several orders of magnitude, this error does not play an appreciable role (6).

For granulocyte adhesion to solid surfaces the following materials were utilized: polytetrafluoroethylene (PTFE), Commercial Plastics, Toronto, Canada; "7sv (S standing for solid and V for vapor) = 17.6 ergs/cm 2 (11); low density polyethylene (LDPE), Commercial Plastics, Toronto, Canada; "/sv = 32.5 ergs/cm 2 (11); polyethylene terephthalate (PET), Celanese, Toronto, Canada; '7sv = 47.0 ergs/cm 2 (11); sulfonated polystY2rene (SPS), Dow Chemical, Midland, Michigan; "Tsv = 66.7 ergs/cm (11). The characteristics of the suspending liquids used are given in Table 1. Liquid surface tensions were measured with the Wilhelmy technique (14).

Granulocyte adhesion experiments were performed with liquid media in which the surface tension was varied by the addition of different amounts of dimethyl sulfoxide (DMSO); see Table 1. Human granulocytes were isolated from whole human blood according to the procedure of BC, yum (15). In brief, the whole human blood mixed with 0.05% (w/v) Na2EDTA

SURFACE TH ERMODYNAMICS OF GRANULOCYTES 117

TABLE l Suspending Liquid Media Used in the Granulocyte Adhesion Determinations

Surface tensions, Liquid medium "/LV, ergs/cm 2

H:O 72.8 HBSS a 72.9 HBSS + 3% b DMSO c 70.5 HBSS + 5% b DMSO 69.4 H20 + 6% b DMSO 68.7 HBSS + 6% ~ DMSO 68.8 H20 + 6% b DMSO + 5% Na2EDTA d 69.0 H20 + 7.5% b DMSO 69.0 HBSS + 7.5% b DMSO 69.0 H20 + 7.5% b DMSO + 5% Na2EDTA 69.1 HBSS + 10% b DMSO 65.5 HBSS + 15% DMSO 63.1

"Hanks Balanced Salt Solution, comprising, in rag/L: anhydr. CaCl2: 140.0; KCl: 400; KHEPO4: 60.0; MgCL2 . 6H20: 100.0; MgSO4 . 7H20: 100.0; NaCl: 8000.0; NaHCO3: 350.0; Na2 HPO4 . 2H20: 60.0; glucose: 1000.0; ~t = 0.15, pH 7.26.

bApproximate concentrations (v/v) of DMSO. Since this material is quite hydrophilic, the apparent concentrations fluctuate considerably as a function of the time the bottle (or vial) has been opened; the important value is the surface tension (YLV), measured just before use (via the Wilhelmy method).

CDimethyl sulfoxide. dThe disodium salt of (ethylene dinitrilo) tetraacetic acid. eThe granulocyte themselves exuded no materials during the duration of the experiments

that lowered or altered the surface tension of the surrounding media (11).

(final concentration) was diluted with physiological saline (1:3). The diluted blood was then layered on top of 3 mL of Lymphoprep (Nyegaard and Co., Oslo, Norway) and centrifuged for l0 min at 400g. After centrifugation the blood cells were separated into two fractions, a white layer at the interface region consisting of mononuclear cells and a bot tom fraction containing erythrocytes and granulocytes. The plasma layer was clear and contained no cells. The latter was first removed and stored. Next the mononuclear cell layer was removed with about half the volume of Lymphoprep. One mL of the Na2EDTA-plasma was added to each tube. Next, 0.4 mL dextran T-500 (4.5% w/v) (Pharmacia, Piscataway, N J) was added to each tube. The contents were then mixed with a Pasteur pipet, and transferred to small tubes with an inner diameter of 8.5 mm; the cell column was about 40 mm high. The tubes were then allowed to stand for approximately 1 h at 4~ during which time the erythrocytes were allowed

118 ABSOLOM ET AL.

to settle. After this period the plasma layer containing the granulocytes was carefully removed to about 1-2 mm above the erythrocyte layer. This was then centrifuged and the cells washed at least five times in Hanks balanced salt solution (HBSS) to remove unwanted plasma proteins. The granulocyte suspension was then counted and adjusted to give a cell count of 1 X 106 granulocytes/mL. Purity of the granulocyte suspension was always > 95%. Viability of the isolated granulocytes was then tested using the trypan blue exclusion test. Viability was always better than 96%.

Adhesion of the granulocytes to the various test materials was performed as described previously (11). Briefly, a 1 mL of a granulocyte suspension containing 1 • 10 6 cells, in whatever test medium, was placed on the surfaces and was retained in wells formed from 2% (w/v) agarose. The cells were then incubated at room temperature for 30 min. Thereafter the surfaces were vigorously washed with HBSS to remove non-adhered cells. Finally, the cells adhering to the surfaces were air dried, and stained using Wright stain. The cells adhering to the various surfaces were then counted using 1000X magnification and corrected to give the number of cells per mm 2.

Results and Discussion

1. Surface Tensions of Granulocytes

In Figs. 1 and 2 the granulocyte adhesion of, respectively, normal control and a patient (G) are plotted versus the surface tensions of the solid materials to which they adhered. The various lines obtained pertain to the different liquid media used. In Fig. 3 these data are plotted under conditions of zero van der Waals, interaction. In Fig. 4, the slopes of the straight lines in Figs. 1 and 2 were evaluated by means of a computer least squares fit and plotted versus the surface tension of the different liquid media used. The surface tensions of the granulocytes, obtained by the intercepts of Fig. 4 (as well as those for the granulocytes of other patients not illustrated here) are given in Table 2, together with the surface tensions found for the same cells via the contact angle method. Clearly, the cell adhesion method yields cell surface tensions that conform closely to those derived through the contact angle method, in the case of normal (11), as well as pathological granulocytes, which considerably strengthens the confidence one may attach to the correlation between the surface tension of cells and their in vitro phagocytic activity (12, 13).

In order to test the validity of the results obtained in Fig. 4 the significance of the difference in the intercepts was determined by means of hypothesis testing. The method employed is briefly described here. The slopes of the straight lines in Figs. 1 and 2 are plotted against the surface tension of the different liquid media used. The surface tensions of the granulocytes were

SURFACE THERMODYNAMICS OF GRANULOCYTES 119

4~

o ,4

oo

z

200

150

IO0

60

�9 HBSs YLV = 7 2 . 9 e r g s / c m 2 O 3% DMSO = 7 0 . 5

�9 5% DNSO = 6 9 , 4

" ~ Zl 7,5% DHS0 : 6 9 . 0

7,5% DZS0 in H20 = 69.1 ~ �9 + 5% EDTA

J - ~'~,~. E] 8% DMSO = 8 8 . 5

~ ' ~ . O 10% DMSO : 6 5 . 5 "

~ �9 15% DMS0 : 63.1 "

[3.

�9

I I I I l 20 30 40 50 60 70

y s v ( e r g s / c m 2 )

FIG. 1. Normal granulocyte adhesion for the DMSO solutions used: �9 Hanks' Balanced Salt Solution (HBSS); O 3.0% (V/V) DMSO in HBSS: �9 5.0% (V/V) DMSO in HBSS; A 7.5% (V/V) DMSO in HBSS; �9 7.5% (V/V) DMSO + 5% (w/v) Na2EDTA in H20; f--I 8.0% (V/V) DMSO in HBSS; O 10.0% (V/V) DMSO in HBSS; �9 15.0% (V/V) DMSO in HBSS. Error limits: 95% confidence (for graphical reasons error limits are not given in all cases). See Table 1 for the surface tensions.

obtained from the intercepts of the curves which were determined from a computer least square fit. The significance of the difference of the surface tensions of the granulocytes of each patient with that of the normal control was calculated using a null hypothesis. The standard errors of the surface tension of the granulocytes were obtained from the sum of the squares of the deviation of the slopes from the value predicted by the curves. Since the number of liquids used was small, a student's t distribution was employed in the hypothesis testing. The surface tension of the granulocytes obtained by the adhesion method are given in Table 2, together with the surface tension found for the same cells using the contact angle method. The significance of the difference in the slope intercept is also given. In the case of the least diseased patient (C) the significance is at a confidence level of 90.0% and in the most diseased patient (G) the significance of the difference is at the level of 99.5% confidence.

200

O HBSS u = 72.9 ergs/cm 2

�9 3% DMSO in HBSS = 70.5

D 5% DMSO : 69.4

A 8% DMS0 = 68.8 "

1 ~ ~ 1601 S~SS~ • ~2B~S :5685:75 "

150

o

i00

120 ABSOLOM ET AL.

�9 " . . . . . . . . . . . . . ~ ' 6 ' I I I x l I ' , 20 30 ~0 50 60 70

Ysv(ergs/cm2)

FIG. 2. Pathological granulocyte adhesion for the DMSO solutions used (Patient G): O HBSS; �9 3% (V/V) DMSO in HBSS; [] 5% (V/V) DMSO in HBSS; /x 6% (V/V) DMSO in HBSS; �9 6%(V/V) DMSO + 5% (w/v) Na2EDTA in H20; [] 10% (V/V) DMSO + HBSS. See Table 1 for the surface tensions. Error limits: 95% confidence level (for graphical reasons error limits are not given in all cases).

The surface tensions found for the granulocytes correlate well with their in vitro phagocytic activity for opsonized Staphylococcus aureus; see Table 2. The granulocytes of the patients studied show decreases of phagocytic activity and increases in contact angle that both are statistically extremely significant. Thus the concomitant decrease in surface tension of patients' granulocytes (derived from the contact angle measurements) is equally significant, even though in actual magnitude the difference (being of the order of only ~ 1%) appears only slight. One must not lose sight, however, of the significance of differences in other physical parameters in biological fluids, where, e.g., a 1% increase in temperature may denote a severe fever, and a 1% decrease in pH an incipient acidosis. Moreover, the real importance of granulocytic surface tensions lies in the value they yield for the interfacial free energy involved in phagocytic engulfment, which in these cases shows interesting differences between normal and pathological granulocytes; see Table 3. The clinical observations and their correlation with cellular surface properties will be published elsewhere in extenso.

SURFACE T H E R M O D Y N A M I C S OF GRANULOCYTES 121

~E E

o

210

150

90

30

----- control

.... patient

�9 o Required DMS0 concentration in HBSS

�9 o Required DMSO concentration in H20

�9 A Required DMS0 concentration

in H20 + 5% Na 2 EDTA

I I 0 o

s

O O

t . . . . . . .

. . . . . . . . . . A K _ . . . . . . . . . . . �9

I "~ A I I 20 qO 60 80

ySV ( e r g s / c m 2 )

FIG. 3. Graphs for the control (broken line) and the patients (solid line) granulocytes when there is an absence of a van der Waals interaction, vs TLV. In the case of the control this occurs when 3' Lv = 69.0 ergs/cm 2, and for Patient G when "yLV = 68.2 ergs/cm 2 (O O).

The effect of the presence of cations on cell adhesion to the substrate is also shown.

l iD-wi thout NaEEDTA ' DMSO in H 20 �9 A -with Na2EDTA + DMSO in H20

It has been noted before (7) that the two major steps in phagocytic engulfment, from the surface thermodynamic point of view, are the steps from incipient adhesion of the bacterium onto the phagocyte to half engulfment, and the step from half to complete engulfment; see Fig. 4. The free energies of these steps can be expressed as follows:

AFengulfment-1 = 1/2 ( T G a - ")/BL)- 1/4 '~/GL [1]

and:

mFengulfment-2 = 1/2 (')/6B - ")/BE) "~ 1/4 ")/GL [2] With normal granulocytes, of which the "YOL is remarkably constant (~- 0.20 ergs / cm2), and different bacteria, with widely varying "yBL (and thus 7oB), it

122

0-8

ABSOLOM ET AL.

0.4

6"4 65 66 67 68 ~ ,~ 69-",..,,. D70 71 72 " "

73

e~

o

1,2

1.6

FIG. 4. Slopes of straight line data of Figs. 1 and 2 vs ~/LV. Slope for control granulocytes is zero for "yLV = "/GV = 69.0 (ergs/cm 2) (broken line); slope for granulocytes of Patient G is zero for "y Lv = "YOv = 68.2 (ergs/cm 2) (solid line); see Table 2.

was found that the phagocytic engulfment increased when mFengulfment-1 (and of course also mFengulfment-2) becomes more strongly negative, which happens when bacteria become more strongly hydrophobic (at values of "/BE ~ 0.25 ergs/cm 2 and up) (7). Thus the more hydrophobic the bacteria, the more pronounced the degree to which they become phagocytized in vitro in aqueous media (e.g., Hanks' balanced salt solution). At a fixed value for 3/OL (constant for all normal human granulocytes) there was hitherto no possibility of distinguishing between the influence of equations [ 1] and [2]. The question now arises whether for one and the same bacterial species, but different granulocytes, there is an equivalent correspondence between in vitro phagocytosis and changes in free energy. The in vitro phagocytic activity is therefore compared with the free energy expression of Eqs. [ 1] and [2] (see Table 3), the latter being calculated both from the contact angles observed on monolayers of the granulocytes as well as from the "y6v values obtained from granulocyte adhesion. We infer that just as in the case of the phagocytosis of various bacteria by one and the same type of granulocyte, the change in free energy is negative in all cases. However, in the present case the mFengulfment-1 actual ly bcomes more negative with increasing hydrophobicity, whereas only z~Fengulfment-2 shows the expected decrease with increasing hydrophobicity of the granulocytes. It thus would appear that the last step in the engulfment process, i.e. the progression from half to complete engulfment, may well be the decisive one.

S U R F A C E T H E R M O D Y N A M I C S O F G R A N U L O C Y T E S 123

l"q

<

o 0

0

o 0

0

t~

zi5

~~

<

0

<

-,~

0

~ ; e g . g �9 - = ~ ~ , ~

~ o ~

o . ~

0 ~-~ ~.~ ~ o~ o p .

0 ~

o

0

r, 0

0

I g ~ o o d d

0 I N r ,~ I ~

g~ ~.. o. o~ o d 7 o b d ~ ~ +1 +1 ~ + 1 " +1 x

+~ +~ V+~ V+~ V O~ oO r O~

" 6 0 ~ o

t..; O

ea "O

+1

z" Z

�9 ~ O

O .

~ + ~

1 2 4 A B S O L O M ET AL.

.1

.<

~ ~ "~ ~.~ ~ o , ~

< " ~ ~ ~

r~ e LS ~

~e~e

~ ~.'~ ~ ~ e

0 ~

e~

o

~ o

~ o ~ o

0 o

o

0

o

o

SURFACE THERMODYNAMICS OF GRANULOCYTES 125

O ~ Fengulfment_ I

FIG. 5. Diagram of two of the major steps involved in particle engulfment m F e n g u l f m e n t - 1 from incipient adhesion to phagocyte surface to first half of engulfment and AFe,gu~fme,t-2 from half to complete engulfment of particle by phagocyte ( 6, 7):

AF..gu,fm..t-1 = 1/2 (VoB - " ) / B L ) - - I/4 VOL AFengulfrnent-2 = 1/2 (VoB - VBL) "3!- 1/4 ")/GL-

The surface tensions yea, '~BL, and ")/GL are derived from the values for y~v and "yav, which in their turn were obtained from contact angle measurements (3/By) and from both contact angle measurements and cell adhesion data ('y~v), via the equation of state approach (8). The contact angle of opsonized Staphylococcus aureus in these experiments was 19.50, corresponding to a surface tension ('yBv) of 68.62 ergs / cm 2.

2. Residual Cell Adhesion in the Absence o f van der Waals Attraction

The horizontal lines in the lower halves of Figs. 1 and 2 represent the degrees of cell adhesion under the condit ion where "yLV = "yGv, i.e., when the net van der Waals at tract ion is zero (16-19). As already noted earlier (I1), there is considerably less cell adhesion at very low or zero (# = 0, in H20) ionic strength than at / . t = 0.15 (in HBSS), which evokes the influence of ionic interactions. To test this possibility further, and because multivalent cations, e.g., Ca 2§ ions, often are implicated in cell adhesion (20), the zero ionic strength liquid medium was also admixed with Na2EDTA, to inactivate any multivalent cations that might still be contributed, for instance through leakage f rom the cells themselves. And indeed, as the lowest horizontal lines in Figs. 1, 2, and 3 indicate, at very low ionic strength and in the absence of plurivalent cations, the adhesion of normal as well as of patients ' granulocytes to surfaces of varying surface tensions is virtually abolished in liquid media, when 3/LV = 3/~v.

Acknowledgment

This work was suppor ted by U S P H S Grant No. N I D R 1 P 50 DE 04898 and by N R C Grant No. 8278. Two of the authors (AWN and DRA) would like to acknowledge the fellowship suppor t of the Ontario Heart Foundat ion .

126 ABSOLOM ET AL.

References

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New York, pp. 176-208. 4. Zisman, W. A., (1963), Ind. Eng. Chem. 55, 18. 5. Zisman, W. A., (1964), Adv. Chem. Ser. 43, 1-51. 6. Adamson, A. W., Physical Chemistry o f Surfaces, 1967, Interscience, New

York. 7, van Oss, C. J., Gillman, C. F., and Neumann, A. W., (1975) Phagocytic

Engulfment and Cell Adhesiveness, Dekker, New York. 8. Gillman, C. F. (1973), Phagocytosis and Cell Adhesiveness as Surface

Phenomena, PhD Dissertation, State University of New York at Buffalo. 9. Neumann, A. W., Good, R. J., Hope, C. J., and Sejpal, M., (1974), J. Colloid

Interface Sci. 49, 291. 10. van Oss, C. J. (1978), Ann. Rev. Microbiol. 32, 19. 11. Neumann, A. W., Absolom, D. R., Zingg, W., and van Oss, C. J., (1979), Cell

Biophys. 1, 79. 12. van Oss, C. J., Bernstein, J. M., Park, B. H., Cianciola, L. J., and Genco, R. J.,

(1979), 6th International Convocation o f Immunology, Buffalo, Milgrom, F., and Albini, B., eds., Karger, Basel, 311-315.

131 Cianciola, L. J., Genco, R. J., Patters, M. R., McKenna, J. and van Oss, C. J., (1977), Nature 265, 445.

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15. BCyum, (1968), Scand. J. Clin. Lab. Invest. 21, Suppl. 97, 77. 16. Neumann, A. W., Omenyi, S. N., and van Oss, C. J., (1979), Colloid Polymer

Sci. 257, 413. 17. van Oss, C. J., Omenyi, S. N., and Neumann, A. W., (1979), Colloid Polymer

Sci. 257, 737. 18. Omenyi, S. N., PhD Dissertation, University of Toronto, 1978. 19. van Oss, C. J., Neumann, A. W., Omenyi, S. N., and Absolom, D. R.,

(1978), Sep. Purif. Methods 7, 245. 20. Curtis, A. S. G. (1962), Biol. Rev. 37, 82.