THE ACTION OF ANTIBODY AND COMPLEMENT ON MAMMALIAN CELLS*

Howard Green and Burton Goldberg Department of Patliology, New York Uninersity-Bellevue Medical Center, New York, N . Y .

The studies discussed in this paper are concerned principally with the action of heterologous antibodies on mammalian cells. Cell-antibody interactions are most conveniently studied in such systems, for it is a simple matter to obtain powerful heterologous antisera. However, the action of homologous antibodies now has been studied 1-4 (see also, C . A. Stetson and E. Jensen, this monograph) sufficiently to make it clear that they produce the same kind of changes in cells as do heterologous antibodies. There is therefore no reason to doubt that the mechanism of action on cells is substantially the same for the two kinds of antibodies.

The effects of antibodies will be described as they are observed in vitro under conditions in which it is possible to distinguish between the effects of antibody alone and those requiring the presence of complement. How these effects may be modified in vivo when the cells are in the form of a solid tissue transplant will not be considered in this paper.

Most studies of the in vitro effects of antibodies on animdl cells have utilized erythrocytes because they represent a homogeneous population of cells uni- formly exposed to the action of substances in the medium. Methods of ob- taining tissue cells in free suspension or in monolayers have provided similar advantages in the use of structurally more complex cells than the anucleate erythrocyte. Although the events occurring in such cells when they are ex- posed to antibody and complement differ in detail from those in the erythrocyte, the mechanism of immune lysis in the two cases appears to be the same.

The Ejects of rl&ibody on Animal Cells in the Absence of Complement When cells in free suspension are treated in vitro with specific antibodies and

studied by phase or light microscopy, agglutination appears to be the major morphologic ~ h a n g e . ~ t 5 An electron microscopic study on the Krebs mouse ascites tumor cell5 has demonstrated that the nucleus, mitochondria, lipid in- clusions, the endoplasmic reticulum and ribosomes were unaffected by antibody action. The cell membrane, however, was distinctively altered by exposure to the antitumor cell gamma globulin. The alteration took the form of a focal series of projections and invaginations of the surface membrane with the corre- sponding production of narrow channels extending into the cytoplasmic matrix. The altered surfaces of apposed cells frequently interdigitated, indicating that the transformation favored agglutination.

It seems possible that this folding of the cell surface could have been brought itbout by normal surface membrane movements (pinocytosis?) bringing sepa- rated antigenic points in the cell membrane into sufficiently close proximity to

* The investigation reported in this paper was supported in part by Research Grants 12-2216 rroni the National Institute of Arthritis and Metabolic Diseases, C-3249 from the National Cancer Institute, SF-319 from the Public Health Service, Bethesda, Md., and by the Lillia liabbitt Hyde Foundation, New York, N. Y.

352

Green 81 Goldberg: Action of Antibody and Complement 353 allow bridging by divalent antibody. Other antibody molecules could then act in “zipper fashion” to fix apposed surfaces. If membrane movements persisted in immediately adjacent zones, the process could extend locally.

Another kind of structural evidence for antigen-antibody binding in cellular membranes has been provided by the electron-microscopic studies of Bessis6 and Rebuck7g8 on erythrocytes exposed to specific blood group antibodies.

The available evidence supports the view that antibody alone does not affect the selective permeability or chemical constitution of cells9 or their metabolic properties.lOJ1 Indeed, mammalian cells can be grown in the presence of sig- nificant concentrations of antibody.12-14 Goldstein and Myrvik12 have prop- agated HeLa cells over 15 subcultures in antibody concentrations 5 to 10 times higher than were necessary to destroy 90 per cent of the cells upon the addition of complement. The growth rate, however, was much below that of the con- trol cultures, and clumping, microcolony formation and some changes in cell shape were noted. Quersin-Thiry13 has grown HeLa cells a t normal rates in antibody concentrations insufficient to cause agglutination, but still capable of protecting the cells against the attachment of polio virus. There is, therefore, considerable evidence that, although antibody combines with antigenic sites in the cell membrane and may impose alterations of surface structure, it does not seriously affect the viability or metabolic functions of the cell.

The Efects of Antibody and Complement on Animal Cells MorfihoZogic changes. In contrast to the relatively innocuous effects of anti-

body alone, the combined actions of antibody and complement are highly lethal to mammalian cells. Morphologically, a variety of cytotoxic changes are noted (see review by Wissler and F1ax,I5 also Goldberg and Green: Goldstein and Myrvik,12 Lumsden,I4 Latta and Kutsakis,16 and Bickis et aZ.ll). Although the details of the structural damage produced may have been modified by the par- ticular in vivo or in vitro conditions employed, most of the studies have indicated that the affected cells become swollen by the entrance of water. With cells in monolayers or in free suspension, the process characteristically begins by the formation of numerous peripheral b l e b ~ , ~ J ~ J ~ J ~ but may finally result in a more or less uniformly swollen cell. Electron microscopic studies of cells in tissue culture18 and in free suspension5 have confirmed the finding that there is a move- ment of fluid into membrane-bounded compartments, including mitochondria, endoplasmic reticulum, and perinuclear space.

At least in the case of antibody-complement treated cells in free suspension, the cell membrane remains unbroken during these events,’t6 but the cells be- come very fragile and can be disrupted easily by mechanical forces.19

The earliest studies were performed on Ehrlich ascites tumor cells by Colter et a1.2O and Ellem;’ r Z 2 and the results were similar to those obtained on Krebs ascites tumor

The earliest and most striking change was the rapid equilibration of small molecules between the cell interior and the medium. K+ was lost from the cells at a rate far greater than its normal leak rate and Na+ passed rapidly into the cells. The cells quickly lost the bulk of their amino and ribonucleotides?‘ s23 The macromolecules of the cytoplasm-RNA and pro -

Chemical changes.

354 Annals New York Academy of Sciences tein---were also 1ost,9,20,23 but a t a slower rate, while desoxyribonucleic acid remained in the cells?,20 The chemical data are consistent with the electron microscopic observation that large numbers of 150A ribonucleoprotein particles (ribosomes) had disappeared from the cytoplasmic matrix of antibody-com- plement-treated Krebs ascites cells: and the bulk of the ribonucleic acid lost from the cells could be recovered as intact ribosomes by centrifugation (105,000 g) of the cell-free medium.lQ Larger structures, however, do not pass into the medium. Thus, in immune cytolysis as in immune hemolysis, leakage of intra- cellular macromolecules occurs through a stretched but morphologically intact cell membrane.

I n view of the large losses of cell constituents from anti- body-complement-treated cells, it is not surprising that metabolic properties of the cells are seriously affected. Substrates, coenzymes, and enzymes are prob- ably lost, although perhaps to varying degrees, depending on the molecular sizes of the substances and their locations in the cell. This could explain the varying susceptibility of different metabolic processes to the action of antibody and complement. For example, the incorporation of CI4 glycine into cell pro- tein is prevented nearly completely by antibody and complement,11z20 and an- erobic glycoly~is ,~~ endogenous respiration,l' and oxygen consumption in the presence of glucoselOsll are abolished. On the other hand, treated cells continue to metabolize succinate, even a t an increased This could be due to ret ention within the cell of the particulate succinic dehydrogenase system of the mitochondria as opposed to the loss of the soluble glycolytic enzymes of the cytoplasm.

Mechanism of the Permeability Alteration Produced by Antibody and Complement The mechanism of production of the observed changes in antibody-comple-

merit.-treated cells is believed to be the following : the normal cell contains a considerably higher protein concentration than the extracellular fluid, but there is normally no osmotic pressure gradient across the cell membrane. This is thought to be accomplished by the action of energy-dependent pumps which, by maintaining the ion concentration inside the cell slightly lower than that outside, compensate for the colloid osmotic pressure gradient. Any condition that leads to equilibration of ions between cell and medium therefore would produce an unbalanced colloid osmotic pressure gradient across the cell mem- brane, and water would flow into the cell. Such a sequence of events has been produced by various means in erythrocytes, where it has been called colloid osmotic hern0lysis.2~

I t was first suggested by Ellem2z that immune lysis might be a form of colloid osmotic lysis, and recent studiesz3 have shown that this is indeed the case. Antibody and complement do cause the equilibration of ions between cell and medium and an osmotic shift of water into the cell. However, the swelling can be prevented in spite of the ionic equilibration by adding bovine serum albumin (BSA) to the medium in a concentration sufficient to balance the rol- loitl osmotic pressure of the cellz3 (H. Green and F. Silverblatt, unpublished ob- servations), Under these conditions, macromolecules (protein and RNA) are not lost from the tumor cells. In antibody-complement-treated erythrocytes

Metabolic changes.

Green & Goldberg: Action of Antibody and Complement 355 the same holds true: K+ is lost from the cells, but hemoglobin is retained when albumin is present in the medium in sufficient con~entration.2~

Therefore, it would appear that the direct action of antibody and complement is to produce “holes” in the cell membrane through which small molecules equi- librate, but osmotic swelling of the cell and stretching of the cell membrane are required for the escape of macromolecules.

Comparative Studies of Osmotic Swelling If Krebs ascites tumor cells are induced to swell by a variety of means, it

can be shown that the loss of macromolecules is not exclusively a function of the degree of swelling. For example, when the cells were incubated a t 0” C., the metabolically dependent pumps failed to maintain the normal cation gra- dients. After a short interval, therefore, cation equilibration occurred, and

Experiment 1 Cold swelling, 6.5

hrs., 0” C .

TABLE 1 A COMPARATIVE STUDY OF OSMOTIC SWELLING IN ASCITES TUMOR CELLS

Experiment 2 Hypotonic swelling, 20

rnin., 37” C.

Packed celi volume

Cell protein (mg.) Cell RNA (mg.)

(mm.3) 10.5 4.25 0.71

30.0 2.86 8.05 18.5 2.30 4.09 0.96 4.32 4.41 1.02 0.72 1.01 0.74 0.71 0.96

Control 1 Expt’l. I %::$ I Control 1 Expt’l. lcontrol Expt’l’’

Experiment 3 Immune swelling, 20

min., 37” C.

Control Expt’l. 24:i I I

~

Methods of preparation of the cells and incubation conditions have been described previ 0us1y.~ BSA was added in EXPERIMENTS 1 and 2 to a final concentration of 0.1 per cent to prevent sticking c f the cells to the tube wall during centrifugation. Packed cell volumes were determined with capillary sedimentation tubes.15 Protein was determined by the method of Lowry et ~ 1 . 2 6 RNA was separated by the method of Schmidt and ThannhauserZ7 and quantitatively measured by the orcinol method of Dische28

colloid osmotic swelling resulted. After about 635 hours, the cells were swollen to almost 3 times normal size, but there was no loss of protein or RNA (TABLE

Tumor cells were also swollen by the slow addition of distilled water to a suspension of the cells in isotonic balanced salt solution (TABLE 1, EXPERIMENT

2). Here too, swelling of the cells to 2.3 times normal volume was not accom- panied by loss of RNA or protein. However, further reduction in the salt concentration of the medium did induce a greater degree of swelling and ap- preciable loss of macromolecules without bursting of the cells (see also EllemZ1).

Included in TABLE 1 for comparison is an experiment in which cell swelling was induced by the action of antibody and complement (EXPERIMENT 3 ) . For a degree of swelling comparable to that of the other experiments, there was abundant loss of both protein and RNA. Therefore, for comparable degrees of stretching, the membrane of an antibody-complement-treated cell was more permeable to macromolecules than a normal cell membrane.

The concept that antibody and complement produce “holes” in the cell mem-

1, EXPERIMENT 1).

356 Annals New York Academy of Sciences brane is useful for the interpretation of differences between immune lysis and other forms of osmotic lysis. Bordet was able to show many years agoz9 that erythrocyte ghosts produced by distilled water lysis could be shrunk by the addition of hypertonic sodium chloride, while those produced by immune lysis could not. Evidently, after release of their hemoglobin, distilled water-lysed erythrocytes again become relatively impermeable to small molecules, while immune-lysed cells remain permeable.

A distinction also may be made with respect to erythrocytes lysed by a va- riety of other agents and immune lysed erythrocytes. Erythrocytes acted on by Clostridium septicum toxin,30 res~rc inol ,~~ salicylate, X ray and ultraviolet irradiation, photodynamic action and other agentsz4 undergo colloid osmotic hemolysis, but the cells may be protected from lysis by sucrose, which is able, as a nonpenetrating solute, to balance the colloid osmotic pressure of hemo- globin and prevent swelling. However, erythrocytes acted upon by antibody and complement are not appreciably protected from hemolysis by sucrose, be- cau5e sucrose is able to enter the cells rapidly through the holes made by anti- body and complement. In this case, in order to prevent lysis of the cells, it is necessary to use a large molecule such as albumin, which cannot penetrate the altered membrane.

Once lesions are produced by antibody and complement in the cell membrane and cation equilibration has occurred, lysis is probably inevitable if the colloid osmotic pressure of the cytoplasm is not balanced by macromolecules in the medium. However, the time required for equilibration and lysis to occur would probably depend upon the number of holes made in the surface membrane. Under conditions where the number of lesions is very small, the time required for the loss of macromolecules might be considerable.

The E jec t s of Molecules of Different Size in Preventing Colloid Osmotic Lys i s of Antibody-Complement-Treated Cells

Colloid osmotic lysis of antibody-complement-treated cells can be prevented by the addition of a substance to the medium only if its molecular size and con- figuration prevent its passage through the altered membrane. By the addition of substances of different molecular weights, a rough estimate could therefore be made of the size of the holes as they are made by antibody and complement. Substances suitable for such a study must have the following properties: (1) they must be very soluble in water; (2) they must be nontoxic to the cell (basic polymers, for example, interact strongly with cells and are very toxic); and (3) they must neither inactivate antibody or complemenl nor interfere with their action on the cell membrane.

The polyvinyl pyrollidones (PVP) satisfied these criteria, at least for the most part. However, they fell short of the ideal in several respects: (1) at high concentrations, they precipitated small quantities of protein from the gamma-globulin and complement solutions; and (2) they were not homogenous products with respect to molecular size, and even their average molecular weights had been determined only approximately. The PVP preparations used in these experiments, K-30 and K-15,* were characterized by viscosity

* General Aniline and Film Corp , New York, N. Y.

Green & Goldberg: Action of Antibody and Complement 357 measurements. An approximate empirical relationship has been established between the Fikentscher values obtained from viscosity measurements and molecular weights; in this way, mean molecular weights of 40,000 and 13,000 have been assigned to K-30 and K-15, respectively, by their manufacturer.

A comparison was made between PVP, BSA, and sucrose as inhibitors of immune hemolysis. FIGURE 1 shows the results of an experiment in which

U M

r

Q, I I I I I I - g 1.00 - -

- 0 0 e:bACA 0

- Q,

O A + L 0.80"- 0

Y 0.60 - 0.40 -

- + 0

0

- .- t 0.20 - 0, L L 0-

- -

Molar concentration x 10' ( PVP or BSA) FIGURE 1. The effect of PVP and BSA on the release of K+ and hemoglobin in immune

hemolysis. Into a series of tubes were placed 1.0 ml. of different concentrations of PVP or BSA, 0.050 ml. of rabbit-immune gamma globulin (66 mg./ml.), 0.050 ml. of washed 50 per cent suspension of mouse erythrocytes, and either 0.20 ml. of complement (fresh rabbit serum) or 0.20 ml. of heat-inactivated complement. After 15 min. incubation a t 37" C., the suspen- sions were centrifuged and the hemoglobin concentrations of the supernatants determined by their optical density a t 541 ms, using the corresponding inactivated complement superna- tant as the blank. The results are expressed as fractions of the total hemoglobin or K+ of the cells released to the medium, Key: A = BSA; 0 = PVP, K-30; 0 = PVP, K-15.

The cell pellets were analyzed for K+ by flame photometry.

3.58 Annals New York Academy of Sciences mouse erythrocytes were incubated in solutions of these substances at various concentrations. Rabbit-immune gamma globulin and complement were added and, after incubation, the amount of hemolysis was determined. I t may be seen that RSA (molecular weight, 65,000) and PVP (K-30) were approximately of equal effectiveness, on a molar basis, in protecting the erythrocytes against immune hemolysis. M hemolysis was 50 per cent inhibited and, a t 3.0 X PVP (K-1.5) was very much less effective in protecting the erythrocytes, a sixfold higher molar concentration being necessary to inhibit hemolysis by 50 per cent. Sucrose was ineffective a t concentrations up to 0.5 M .

I t will be noted that there was virtually complete release of K+ from the antibody-complement-treated cells a t all the PVP and BSA concentrations shown. I t is therefore clear that the protection against hemolysis afforded by BSA and I’VP was not due to inhibition of the action of complement or anti- body. However, at concentrations higher than those shown here, both PVP and BSA can diminish the loss of K+ as well. This might be due to interfer- ence with the action of antibody and complement, although other explanations are also possible.

A similar study of the ability of PVP, BSA, and sucrose to inhibit the swell- ing of antibody-complement-treated Krebs ascites tumor cells was based on comparisons of packed cell volumes after incubation of the suspensions. The results showed a very similar pattern over the same concentration ranges shown in FIGURE 1. PVP i:K-30) and RSA were equally effective, I’VP (K-15) con- siderably less effective, and sucrose virtually ineffective (F. Silverblatt and H. Green, unpublished observations).

I t appears from these data that the holes made in the cell membrane by the action of antibody and complement were sufficiently large to admit a signifi- cant part of the low molecular-weight PVP preparation, but not substances of molecular weight range 40,000 to 65,000. Precise determination of the hole size was prevented by the fact that the PVP preparations were not homogene- ous, and also by the short time (15 min.) allotted for equilibration. As the fit bet ween hole size and molecule size becomes closer, the time required for equili- bration would become longer.

At concentrations of 1.8 X M , completely inhibited.

Location of fhe Antigens Responsible for Immuae Cyfotoxicity The occurrence of indifferent antigen-antibody reactions in close proximity

to, or even perhaps within the ascites tumor cells, apparently has no effect on the cells. Tumor cells were incubated in the presence of various proteins for intervals of several hours, and the suspensions were then injected into mice immunized by a series of injections of the respective proteins in Freund’s ad- juvant. Incubations were also performed in the presence of heat-denatured proteins, for it has been shown that such proteins are taken up by the cells a t greatly increased rates (H. Green, unpublished observations). In all cases, the cells grew out as rapidly in mice immunized against the specific proteins as cells incubated for the same interval in the absence of the protein and injected into nonimmune mice. Also, incubation of the cells first in antigen, then in rabbit antiserum to the antigen,

Some of the combinations used are shown in TABLE 2 .

Green & Goldberg: Action of Antibody and Complement 359 did not delay the growth of the tumor cells when they were subsequently in- jected into mice.

These results are not surprising, for in all probability only the combination of certain cellular antigens with their respective antibodies and complement results in permeability changes. Since all the results discussed earlier point to the cell membrane as the site of action of antibody and complement, it would seem reasonable that membrane antigens are responsible. The results of studies on the cytotoxic effects of antibodies produced by immunization with cell fractions are probably not incompatible with this view. Antibodies pre- pared in rabbits against soluble proteins of mouse ascites tumor cells were shown by Colter et al. to be ineffective in damaging the tumor cells as judged by their failure to inhibit cell or to produce ioss of cell protein and RNA.2O Antibodies produced against ribonucleoprotein and desoxyribonu- cleoprotein fractions, however, were effective but one wonders if the prep- arations did not contain membrane antigens.

TABLE 2

Cells incubated in

Human serum albumin Heated (80” C . , 60 min.) human serum albumin Limulus hemocyanin Sulfanilic acid azo hemocyanin Bovine gamma globulin Sulfanilic acid azo bovine gamma globulin Sulfanilic acid azo human fibrinogen

Protein conc. (ms./ml.)

0.085 0.085

j I 1.0

Incubation time (min.)

180 180 225 225 225 225 225

.-

Conclusions

Recent studies on the action of antibodies and complement on mammalian cells in vitro permit the following conclusions:

Specific antibodies may alter the ultrastructure of the cell membrane, but the permeability properties and metabolism of the cells remain unaffected. Mammalian cells can remain viable in the presence of appreciable concen- trations of antibody.

Antibody and complement are both required to produce irreversible cell damage, and the cell membrane appears to be the site of their action. A leakiness in the cell membrane is induced that permits the loss of low mo- lecular weight constituents and the equilibration of cations between the cell and the surrounding medium. The equilibration leads inevitably to the en- trance of water into the cell along a colloid osmotic pressure gradient. The cell is expanded, the cell membrane stretched, and “holes” of sufficient size are made in the membrane to permit the escape of macromolecules into the medium.

These events represent a form of osmotic lysis, but immune lysis differs from osmotic lysis induced in other ways in the greater degree of leakiness produced in the cell membrane. The cell membrane exposed to antibody and complement is readily permeable to molecules of the size of sucrose and larger

3 60 Annals New York Academy of Sciences and, for comparable degrees of osmotic stretching, only the immune damaged membrane allows macromolecules to escape from the cell.

References 1. KALFAYAN, B. & J. G. KIDD. 1953. Structural changes produced in Brown-Pearce

J. Exptl. Med. 97:

The cytotoxic activity of isoantibodies in mice.

Toxicity of homologous immune serum to a J. Natl.

1959. Antibodv resDonse to homo-

carcinoma cells by means of a specific antibody and complement.

Transplantation Bull. 3: 142-143.

transplantable tumor: studies using phase microscopy and cinemicrography. Cancer Inst. 16: 1021-1045.

145-163. 2. GORER, P. A. & P. O'GORMAN.

3. SCHREK, R. & F. W. PRESTON.

1956.

1956.

4. 'rERASAKI. P. I.. T. A . CANNON & W. P. LONGMIRE.

5.

6. 7.

8.

' I .

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

2 L

grafts. 'Proc: Soc. Exptl. Biol. Med. 102: 280-284.

and complement on Krebs ascites tumor cells. Med. 109: 505-510.

GOLDBERG, B. & H. GREEN. 1959. The cytotoxic action of immune gamma globulin J. Exptl.

BESSIS, M. 1950. Studies in electron microscopy of blood cells. Blood. 6: 1083-1098. IZXBUCK, J. W. Structural changes in sensitized human erythrocytes observed

tZmucK, J. W. 1959. In Mechanisms of Hypersensitivity. Henry Ford Hospital,

GREEN, H., R . A. FLEISCHER, P. BARROW & B. GOLDBERG. 1959. The cytotoxic action 11. Chemi-

J. Exptl. Med. 109: 511-521. FLAX, M. H. 1956. The action of anti-Ehrlich ascites tumor antibody. Cancer Re-

BIcKis, I. J., J. H. QUASTEL & S. I. VAS. Effects of Ehrlich ascites antisera on Cancer Research.

GOLDSTEIN, G. & Q N. MYRVIK. 1958. The reversible and irreversible toxic effects J. Immunol.

QuERSIN-THIRY, L . 1958. Action of anticellular sera on virus infections. J. Immunol.

LUMSDEN, C . E. 1959. Effects of antibodies on cells in tissue culture. I n Immuno- pathology, 1st Intern. Symposium. : 262-277. P. Grabar and P. Miescher, Eds. Benno Schwahe. Basel. Switzerland.

I. Ultrastructural studies.

1953. with the electron microscope.

Intern. Symposium. : 138-141. Little, Brown. Boston, Mass.

of immune gamma globulin and complement on Krebs ascites tumor cells. cal studies.

search. 16: 774-783.

the biochemical activities of Ehrlich ascites carcinoma cells in vitro. 19: 602-607.

Anat. Record. 116: 591-613.

1959.

of anti-HeLa cell I-abbit serum upon human cell lines in tissue culture. 80: 100-105.

81: 253-260.

WISSLER, K. W. & M. H. FLAX. 1957. Cytotoxic effects of antitumor serum. Ann.

LATTA, H. & A. KUTSAKIS. Cytotoxic effects of specific antiserum and 17-hy-

MILLER. D. G. & T. C. Hsu. 1956. The action of cvtotoxic antisera on the HeLa

N. Y . Acad. Sci. 69(4): 773-794.

droxycorticosterone on cells in tissue culture. 1957.

Lab. Inv. 6: 12-27.

strain of human carcinoma.

microscopy.

press.

studies with antisera against tumor cell protein fractions. 276.

Australian J. Sci. 20: 11G117.

ogous antiserum.

on permeability control in ascites tumor cells and erythrocytes.

Helv. Physiol. Acta. 6: 234-246.

Cancer Research. 16: 3061-312. LATTA, H. A cellular reaction to antibody in tissue culture studied with electron

GOLDBERG, B. & H. GREEN. Immune cytolysis. J. Biophys. Biochem. Cytol. In

Cancer Research. 17: 272-

Studies on the mechanism of the cytotoxic action of antisera.

Some aspects of the ascites tumor cell response to a heterol-

Effect of antibody and complement J. Exptl. Med. 110:

WILBRANDT, W. 1948. Der kompensationstest der kolloidosmotischen hamolyse.

VAN ALLEN, C. M. 1926. Volume measurement of blood platelets. J. Lab. Clin. Med.

LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR & R. J. RANDALL. 1951. Protein meas-

1959. J. Biophys. Biochem. Cytol. 6: 405-410.

COLTER, J. S., D. KRITCHEVSKY, H. H . BIRD & R. F. J. MCCANDLESS. 1957. I n &VO

ELLEM, K. A. 0. 1957.

ELLEM, K. A. 0. 1058.

GREEN, H., P. BARROW & B. GOLDBERG. Cancer Research. 18: 1179-1185.

1959.

699-7 13.

12: 282-285.

urement with the Fohn phenol reagent. J. Biol. Chem. 193: 265-275.

Green & Goldberg: Action of Antibody and Complement 361 27. SCHMIDT, G. & S. J. THANNHAUSER. A method for the determination of desoxy-

J. Biol.

28. DISCHE, Z. 1955. In The Nucleic Acids. 1: 3W302. E. Chargaff and J. N. Davidson,

29. BORDET, J. 1939. Trait6 de l’Immunit6. 2nd ed. : 370. Masson. Paris, France. 30. BERNHEIMER, A. W. Comparative kinetics of hemolysis induced by bacterial

31. LATTA, H. & S. S. POLT. 1954. Sucrose inhibition of resorcinol hemolysis. Science.

32. COLTER, J. S., H. KOPROWSKI, H. H. BIRD & K. PFEISTER. 1956. Immunological Nature.

1945. ribonucleic acid, ribonucleic acid and phosphoproteins in animal tissues. Chem. 161: 83-89.

Eds. Academic Press. New York, N. Y.

1947. and other hemolysins. J. Gen. Physiol. 30: 337-353.

120: 271-273.

studies with protein fractions isolated from Ehrlich ascites carcinoma cells. 177: 994-995.

Discussion of the Paper

LAWRENCE: Green and Goldberg have done something in an area that has provoked many who are concerned with hypersensitivity on the one hand and with the behavior of microbial cells on the other. They have adapted the tech- niquesof Monod and his school to mammalian systems and, in a slower-growing cell population, have clarified the events that occur in the membranes of sensi- tized cells. I think this would also be the system par excellence to demonstrate an effect of a delayed allergen such as tuberculin on tuberculin-sensitive cells.

Some work with which Pappenheimer and I were concerned has a bearing on this. If leukocytes from tuberculin-sensitive individuals are incubated with antigen, the transfer factor activity appears in the supernatant and the cells are then desensitized. When these cells are spun out and examined by the usual methods, they appear to be intact and viable. I wonder what a system such as Green and Goldberg have introduced here might have revealed?

ZOLTAN OVARY: Until now very little attention has been given to what hap- pens to the cell when all components of complement C’ were fixed.

The work of Mayer and his collaborators, and also of others, established very clearly the sequence and the kinetics by which different components of C’ fix on the sensitized erythrocyte in immune hemolysis. We know that when C’ reacts with the sensitized erythrocyte, the first to be fixed is the first component and then comes the fourth component. These steps require cations. Only when these two components have reacted can the second component get fixed, and this step requires magnesium ions. Finally, the two constituents of the third component react, and then the erythrocyte is in a state designated E STAR. For these last steps no cations are required, The reaction of the components of C’3, however, is more temperature-dependent than the former steps. It was believed until now that nothing can prevent the lysis of a cell in a state of E STAR. I t was not well understood, however, why not all the E-STAR cells undergo immediate lysis. The experiments of Green and Goldberg explain per- fectly what happens. The time for lysis would depend on the number of holes made in the membrane which, in turn, determines the rate of cation equilibra- tion and consequent swelling.

These experiments also give a clue to what happens in vivo when cytotoxic antibody is injected. For example, it was shown long ago that when anti- Forssman antibody is injected into the guinea pig, an animal with the Forssman

362 Annals New York Academy of Sciences hapten in its cell, necrosis and death of the cell occurs. There is good evidence indicating that the action of this cytotoxic antibody leads C’ to cause necrosis and death of the cell.

In homotrans- plantation immunity, if cytotoxic antibody plays a role, as many believe it does, the mechanism may be the same as that described by Green and Goldberg.

It would be moit interesting to know what happens to the cell if swelling and the outflow of proteins are prevented. Does it remain viable? Do the processec, of metab- o1i.m continue in such a cell?

With homologous antibody the pattern must be the same.

A new field of investigation is opened by these experiments.

Can it heal?

HOWAKD GREEN: We have incubated these cells in the presence of a variety of proteins having nothing to do with the celts, that is, with pure proteins such as serum albumin and bovine gamma globulin for various periods of time; then we injected the celk into mice immunized against these proteins. We found that this had no effect at all on the ability of these cells to grow out in these mice.

With respect to bacteria, of course, the colloid osmotic pressure gradient be- tween the inside of the cell and the outside is supported by a rigid cellular mem- 1)rane: a rigid wall. When this wall is removed by any of a variety of known means, these bacteria must swell in the same way that animal cells swell. No mechanism of this sort exists in animal cells in which the cellular membrane is a purely passive structure and in which the colloid osmotic pressure gradicnt is compensated for by completely different means.

As to Ovary’s questions: we have tried to see whether cells treated with anti- body and complement in the presence of protective concentrations of protein might, if they were Icft in such protein for a long time, recover in some way, but thus far we find no evidence of their recovery.

These cells are nonviable when they are injected into mice, and they do not recover the state of cation impermeability that they had before being exposed to antibody and complement.