differences in the mechanism in vitro immune hemolysis related...

Journal of Clinical InvestigationVol. 43, No. 5, 1964

Differences in the Mechanism of In Vitro Immune HemolysisRelated to Antibody Specificity *

DAVID A. SEARS,t ROBERTI. WEED,t AND SCOTT N. SWISHER§ WITH THE TECH-XNICAL ASSISTANCE OF NORMATRABOLD

(From the Departments of Medicine and Radiation Biology, University of Rochester Schoolof Medicine and Dentistry, Rochester, N. Y.)

This investigation of the mechanism of immunehemolysis was undertaken to define qualitativelyand quantitatively the processes involved in thebreakdown of the human red cell membrane pro-duced by the action of complement (C') andhemolytic antibodies of differing specificity.

Recent investigations in man and animals haveprovided insight into patterns of immune hemo-lysis occurring in the intact organism (1-4). Atthe cellular level, the kinetics of antibody de-pendent hemolysis and of C' action have beenstudied extensively by many investigators, uti-lizing the classical sheep erythrocyte-rabbit anti-body-guinea pig C' system (5). Ponder (6) hasdescribed the kinetics of hemolysis produced by awide variety of chemical and physical agents.Little is known, however, about the nature of theinjury to the human red cell membrane induced byantibody and C' and the mechanism by which thatinjury ultimately leads to hemolysis.

Although the normal intact red blood cell isisotonic with its plasma environment, as recentlydemonstrated by Williams, Fordham, Hollander,and Welt (7), this is possible only because of aneffective or net impermeability to cations that,

* Submitted for publication November 18, 1963; ac-cepted January 15, 1964.

Supported in part by the U. S. Atomic Energy Com-mission at the University of Rochester Atomic EnergyProject, Rochester, N. Y.

Presented in part at the 55th Annual Meeting, TheAmerican Society for Clinical Investigation, AtlanticCity, N. J., April 28, 1963.

t Trainee under training grant 5-TI-AM 5113-08NIAMD, National Institutes of Health. Present address:Walter Reed Army Institute of Research, Washington,D. C.

$ Recipient of U. S. Public Health Service researchgrant HE-06241-02.

§ Recipient of research grant AM-00537-15 from theNational Institute of Arthritis and Metabolic Diseases.

to preserve electrical neutrality, permit no netpassage of electrolytes, but anions, hydrogen, andhydroxyl ions can rearrange themselves. How-ever, the disruption of this normal cation im-permeability permitting free diffusion of cationsacross the membrane, because of the Donnan ef-fect, leads to cell swelling and eventual cell lysis,i.e., escape of hemoglobin. This mechanism ofhemolysis was described in 1936 by Davson (8)and was termed colloid-osmotic hemolysis byWilbrandt in 1941 (9). Recently, Dick has sum-marized the topic clearly (10). An analogoussystem has been demonstrated by Goldberg,Green, Barrow, and Fleischer (11-13) in theirstudies of the lysis of Krebs ascites tumor cellsby rabbit antibody and C'. The cells exposed toantibody and C' rapidly became permeable tocations and other small molecules. Cell swellingand escape of larger molecules such as RNAwere prevented by increasing the osmotic pressureof the external medium with added albumin.Since sucrose did not prevent cell swelling or lossof the large molecules of the cell, these authorsconcluded that antibody and C' had created func-tional "holes" in the cell membrane of a size be-tween that of sucrose and albumin and that os-motic swelling was necessary to permit the egressof large molecules from the damaged cells. Basedon these observations and the finding of a similarmechanism in antibody-C' lysis of mouse erythro-cytes, Green and Goldberg (14) have suggestedthat colloid osmotic lysis is a general mechanismby which animal cells injured by antibodies and C'are lysed.

Our investigation was designed to study anti-body-C' hemolysis of human red cells in vitroutilizing methods similar to those employed byGreen and associates (13) to determine if anti-body-C' hemolysis proceeded by a colloid-osmotic

975

DAVID A. SEARS, ROBERTI. WEED, AND SCOTT N. SWISHER

mechanism in this situation and to quantitatethe size of induced membrane defects. If the ac-tion of antibody and C' produces membrane de-fects too small to permit the direct egress ofhemoglobin, cell swelling and hemolysis should beprevented by the addition to the medium of non-penetrating and, therefore, osmotically activemolecules in appropriate concentrations to balancethe greater intracellular osmotic pressure. Sinceonly molecules larger than the membrane defectremain outside the cell and thus prevent cellswelling and hemolysis, an estimate of the sizeof the membrane defect can be obtained by testingmolecules of various known sizes in the medium.In the present study, we have investigated theeffects of sucrose, bovine serum albumin, humanhemoglobin, and dextran fractions of variousmolecular sizes on in vitro hemolysis of humanred cells by three different antisera and C'.

Methods and MaterialsRed blood cells. Blood was obtained f rom hemato-

logically normal adult male and female donors of groupsA, B, AB, and 0; both Rh-positive and -negative donorswere included. A fraction of blood was mixed with ap-proximately 1 U of heparin per ml of blood, and thesample was centrifuged at 1,700 X g for 10 minutes.The plasma and buffy coat were removed by aspirationand the red cells washed three times with 5 vol of 1%sodium chloride.

Complement. A second sample of the same blood fromwhich red cells were obtained was allowed to clot for 1hour at room temperature. The serum was removedand utilized as a source of C'.

Rabbit antihuman red cell seruom. Blood was collectedin standard acidified citrate dextrose from a type 0,Rh.+, NN human donor. The red cells were separatedand washed four times with 3 vol of 0.9%o sodium chlo-ride. One ml of packed red cells was injected intra-venously into each of four 2- to 3-kg adult rabbits.Similar injections were repeated twice at 10-day inter-vals. Eight and 11 days after the third immunization,the rabbits were bled from ear veins, and serum was col-lected. The sera from the four rabbits were pooled andheated for 30 minutes at 560 C, Seitz-filtered, andstored in the frozen state at - 20° C. To ascertain theantibody titer of this serum, fresh, washed human redcells of groups A, B, AB, and 0 were prepared as 3%suspensions in f resh autologous serum. Portions ofthese red cell suspensions were incubated for 1 hour at370 C with saline dilutions of the pooled rabbit serum.Hemolysis and direct agglutination were then observed,and, in tubes beyond the agglutination end point, anti-globulin tests were performed on those tests involvinghuman A + erythrocytes. A sheep anti-rabbit globulin

serum, from which the heteroagglutinin for human redcells had been removed by repeated adsorption withgroups A, B, and 0 cells at 4° C and at room tempera-ture, was employed in a single dilution. The titers thusobtained for the rabbit antiserum against human groupA cells (both A, and A2) were as follows: hemolysis,1: 100; direct agglutination, 1: 4,000; and positive anti-globulin test, 1: 16,000. Similar agglutinin and hemolysintiters were observed with the other test erythrocytes.

Human anti-A serum. Whole serum containing a hightiter of human anti-A of "immune" character had beenobtained from a group 0 individual (15) previously andstored frozen at - 200 C. The serum was filteredthrough a Millipore filter 1 (0.2 mju pore size) to re-move a small amount of particulate matter. Titers ofthis serum against the group A, red cells employed inthese experiments were as follows: hemolysis, 1: 64;direct agglutination, 1: 2,048. The titers against humanA. cells were somewhat lower, and A, cells were usedin all experiments with this antiserum.

Rabbit antihuman A serum.2 The serum was heatedat 56° C for 20 minutes and absorbed twice with guineapig kidney to remove anti-Forssman antibody. The ab-sorbed serum was titered against human group A, redcells with the following results: hemolysis, 1: 40; directagglutination, 1: 5,120.

Fractionzation. of antisera. One-ml samples of the rab-bit antihuman red cell serum and the human anti-A weredialyzed against 0.02 M phosphate buffer (pH 6.31) at 40C for 24 hours. The dialysates were applied to smalldiethylaminoethanol (DEAE) cellulose columns to sepa-rate 7 S, y2 f rom 19 S, ym globulins as described byKochwa, Rosenfield, Tallal, and Wasserman (17). The7 S globulins were eluted from the column with the 0.02M phosphate buffer and the 19 S with 1 M sodium chlo-ride, both at a flow rate of 1 drop per 20 seconds.

To concentrate the activity of the antibody, the 7 Sfractions were mixed with an equal volume of saturatedammonium sulfate solution. The precipitate was allowedto develop for 30 minutes at room temperature and wasthen removed by centrifugation and redissolved in a smallvolume of water. The resulting solution was dialyzedfor 24 hours against 0.9% sodium chloride at 40 C.Isotonic solutions of the 7 S fraction of human anti-Awere thus obtained with titers of hemolytic activityagainst human group A, red cells only slightly less thanthose of the whole serum. The fractions eluted fromthe column with 1 M sodium chloride exhibited primarilyagglutinating activity and no demonstrable hemolyticactivity against human group A, cells.

Hemoglobin solution. Human red cells were washedthree times with 1% sodium chloride and hemolyzed with3 vol of distilled water. After centrifugation of 12,000X g for 45 minutes, the clear supernatant hemoglobin

1 Millipore Filter Corp., Bedford, Mass.2 Kindly provided by Dr. Harold Baer, Division of

Biologics Standards, National Institutes of Health [de-scribed by him as "pool l" (16)1.

976

MECHANISMOF IMMUNEHEMOLYSISAND ANTIBODY SPECIFICITY

solution was removed and dialyzed against hypotonic so-

dium chloride solution at 4° C for 32 hours. After con-

centration by pervaporation, a final solution of 3.7 mMhemoglobin in isotonic sodium chloride was obtained thatcontained only 1 mEq per L of potassium ion.

Albumin. Commercial bovine serum albumin (Frac-tion V)3 was used; the generally accepted molecularweight of 69,000 was assumed.

Dextrans. Well-characterized dextran fractions ofdifferent molecular weights were utilized.4 Numberaverage molecular weights of the polydisperse dextranfractions were used for calculations of dextran osmolar-ity, since these measurements best reflect their colliga-tive properties, as described by Billmeyer (18).

Preparation of samples and incubation. When albuminand sucrose were used to increase the osmotic pressure

of the medium, these substances were dissolved directlyin the fresh serum at the desired concentrations. Thedextrans could not be dissolved in unheated serum insufficient concentration. These were added to serum as

concentrated solutions dissolved in 0.1 M Tris buffer(pH 6.4) adjusted to isotonicity with sodium chloride.Warming of the dextran and buffer in a water bathexpedited solution. In the experiment utilizing hemo-globin in the medium, the above described hemoglobinsolution in isotonic sodium chloride was added to serum.

In all experiments, the pH of the suspending mediumwas adjusted to 7.0 to 7.2 with HCl before incubation.The desired volume of red blood cells then was addedto the suspending medium. Finally the antiserum was

added. The relative proportions of antiserum, fresh se-

rum, and red blood cells were kept constant in each ex-

periment, and appropriate control samples lacking anti-serum or containing C' inactivated by heat (560 C for 30minutes) were included in each protocol. The totalvolume of test samples varied in different experimentsfrom 2.0 to 4.4 ml and the hematocrits of incubationmixtures from 2.3 to 10 vol per 100 ml. Samples were

incubated under air in siliconized flasks for 2 hours at370 C in a Dubnoff metabolic shaker. In all experi-ments the percentage of hemoglobin and potassium lostfrom the red cells was quantitated after -incubation.

Determination of hemoglobin lost from incubated redcells. Hemoglobin determinations by the cyanmethemo-globin method of Crosby, Munn, and Furth (19) were

carried out on the whole sample and the supernatant me-

dium after incubation. Hematocrits were determined bythe microhematocrit method. The percentage of hemo-globin loss from the erythrocytes was then calculated bythe following formula: OD supernatant medium X (100- hematocrit)/OD whole sample X dilution factor. Evenin experiments in which no protective macromoleculeswere added to the medium, the percentage of loss of hemo-globin from the cells in each sample was somewhat lessthan the percentage of loss of potassium. This differ-

3 Obtained from Squibb and Co., New York, N. Y.4 Obtained from Pharmacia Fine Chemicals, Inc., Upp-

sala, Sweden, and Rochester, Minn.

ence was accounted for by the retention of some hemo-globin by the lysed red cells. Hemoglobin retention byred cell ghosts is well recognized and has been describedby Ponder (20) and Passow and Tillman (21). In thepresent experiments, determinations of the hemoglobincontent of ghosts resulting from the lysis of red cells bythe rabbit antihuman red cell antiserum confirmed the re-

tention of hemoglobin by lysed cells. In the absence ofprotective additives in the medium, this difference betweenpotassium and hemoglobin loss was generally less than10%.

In one series of experiments in which hemoglobin it-self was added to the medium as a protective macro-

molecule, the percentage of loss of hemoglobin from thered cells was determined in a different manner. Thetest red cells were incubated for 90 minutes at room

temperature with sodium chromate-Cr5" and were thenwashed three times with 1% sodium chloride. The radio-activities of the red cells thus labeled and of the super-

natant fluid of each sample after incubation were com-

pared in a well-type scintillation counter. The percent-age of the red cell radioactivity appearing in the super-

natant liquid after incubation permitted estimation of thepercentage of hemoglobin lost from the cells. Prelimi-nary studies had established that Cr51, which becomestightly bound to intracellular hemoglobin, is not releasedto competitive binding by hemoglobin added to the me-

dium.Potassium determinations. Potassium was determined

in samples and standard solutions by flame photometry5utilizing an internal lithium standard. The percentageof red cell potassium lost to the medium during incuba-tion was calculated from the potassium content of the redcells before incubation, that of the supernatant fluid be-fore and after incubation, and that of the sample volumeand the hematocrit after incubation.

In some experiments, the osmotic fragility of the un-

hemolyzed red cells was determined after incubation bythe method of Parpart and colleagues (22). Red cellsand red cell ghosts were quantitated by counting witha Coulter model A electronic particle counter.6 Ultra-filtration was performed according to the method ofToribara (23).

Osmotic pressure measurements. The concentration,and hence osmotic pressure, of hemoglobin in the cell is

much greater than the concentration of protein in serum.

In these experiments the addition of macromolecules to

the medium was designed to balance this greater osmoticpressure and thus to prevent cell swelling. The concen-

trations of albumin and other macromolecules used were

selected both on the basis of published data on the os-

molarity of hemoglobin and the osmolarity of serum al-bumin, as well as on the results of some direct measure-

ments of osmolarity. Osmotic pressure measurementson concentrated albumin solutions have been reported by

5 Baird-Atomic flame photometer, Baird Atomic, Inc.,Cambridge, Mass.

6 Coulter Electronics, Hialeah, Fla.

977


Scatchard, Batchelder, and Brown (24) and on concen-trated hemoglobin solutions by Adair (25). Comparisonof these data indicates that the concentrations of albuminused in these experiments were adequate to balance theosmotic pressure exerted by the intracellular hemoglobin.

Total osmolarity measurements were performed witha vapor pressure osmometer 7 standardized with sodiumchloride solutions. When the osmotic pressure of normalhuman serum was compared with that of serum to which2.6 mM albumin had been added, an increase of 38mOsmper L was observed. However, ultrafiltration ofa solution of the bovine serum albumin in deionized waterand subsequent osmotic pressure measurements on theultrafiltrate demonstrated that such solutions contained20 mOsmper L of ultrafiltrable nonprotein material ofwhich approximately 10% was found to be sodium chlo-ride. Since the ultrafiltrable material could be expectedto cross altered, cation-permeable red cell membranes,however, the effective increment in osmotic pressure dueto added macromolecules in a 2.6 mMsolution of thisalbumin in serum was 18 mOsmper L. This figure isin good agreement with that of Scatchard and co-work-ers (24) and is comparable to the osmotic pressure ofintracellular hemoglobin as discussed by Adair (25).Since the ultrafiltrable material would not be expectedto offer protection, it was not removed. Although ade-quate centrifugation of cells from 2.6 mMadded albuminis not possible, measurement of the volume of normalcells in 2.0 mMadded albumin reveals no significantchange in cell volume.

The osmotic coefficient of dextran, like that of albuminand hemoglobin, has been shown by Rowe (26) andGr6nwall and Ingelman (27) to rise disproportionatelywith increasing concentration in solution. Osmotic pres-sure measurements on a 3.2 mMsolution of Dextran 40in normal serum showed an increase in osmotic pressureof 14 mOsmper L over serum without dextran.

Results

Studies utilizing rabbit antihuman red cellserum. Preliminary experiments showed thatwhen group Al human red cells were incubatedwith rabbit antihuman red cell serum and freshautologous serum as a source of C', the rate ofhemolysis increased with increasing amounts ofantiserum per volume of red cells. The percent-age of red cell potassium lost during incubationparalleled the percentage of hemoglobin lost.After studies of the time course of hemolysis bythis system, 2-hour incubations were chosen sincewe had found that at that time the rate of hemo-lysis was nearly constant. Red cells incubatedwithout antiserum showed negligible loss of redcell potassium and hemoglobin in 2-hour incuba-

7Mechrolab Inc., Mountain View, Calif.

tions. Similarly, the red cells in samples con-taining heat-inactivated C', in spite of markederythrocyte agglutination after incubation, lostonly a minimal proportion of their potassium orhemoglobin, generally well less than 10%.

The addition of albumin to the medium broughtabout a marked change in the pattern of loss ofcellular contents (Figure 1). Potassium lossfrom the red cells was unaltered, but hemoglobinloss was greatly inhibited. With 3.9 mMalbuminadded to the medium,8 72% of the red cell potas-sium was lost but only 5% of the hemoglobin.Antibody and C' had altered the cation perme-ability of the cell membrane to allow potassiumescape. Hemolysis had been prevented because ofthe osmotic effect of the added albumin, whichbalanced the osmotic pressure of the macromole-cules in the cell and prevented cell swelling.

To substantiate this interpretation further, wedetermined the osmotic fragility of cells thus"protected" by albumin against hemolysis. Dur-ing 2 hours of incubation only 19% of the hemo-globin escaped to the medium, whereas 91 % ofthe red cell potassium was lost. The osmoticfragility of these cells (Figure 2) showed that

8 The albumin concentrations refer to the amountsadded to the medium and do not include the concentrationof albumin, or other macromolecules, already present inthe serum.

100

80- \

40- \

ck:z

ALBUMIN CONC. ADDEDTO MEDIUM(MILLI MOLAR)

FIG. 1. THE EFFECT OF ALBUMIN ON LOSS OF POTAS-SIUM AND HEMOGLOBINPRODUCEDBY RABBIT ANTIHUMANRED CELL ANTISERUMAND C'. Each sample contained 0.2ml red cells, 1.6 ml fresh serum containing the added al-bumin, 0.1 ml antiserum, and 0.1 ml 1%o sodium chloride.

g78


fiftyfold dilution of the medium with isotonic(1%o) saline resulted in lysis of 84%o of the cells.Control samples incubated without antibody orwith C' inactivated showed essentially normalosmotic fragility. Thus, the cells incubated withantibody in the presence of albumin clearly hadbecome permeable to sodium as well as to po-tassium.

When Dextran 40 (weight average molecularweight, 35,700) was added to the medium, theresults were similar to those obtained with al-bumin (Figure 3). L'bss of potassium was un-affected, but loss of hemoglobin was almost totallyinhibited by the higher concentrations of dextran.As in the experiments with albumin, dilution ofthe medium of the dextran-protected red cellswith 1% saline for osmotic fragility determina-tions resulted in marked hemolysis; the extentof the hemolysis again agreed closely with theamount of potassium lost before dilution.

To define the lower limit of size of moleculesthat could protect against loss of hemoglobin,Dextran 10 (weight average molecular weight,9,400), Dextran 20 (weight average molecularweight, 22,700), and sucrose (molecular weight,342) were added to the medium in similar ex-periments. Figure 4 illustrates the results ob-tained utilizing Dextran 10. Obviously Dextran10 provides no protection against loss of hemo-globin from the red cells injured by antibodyand C'. Figure 5 illustrates that even when

100'

t 80

60

14040-

SLn

1.00 0.90 0.80 070 0.60 050 0.40

PERCENTSODIUM CHLORIDE CONCENTRATION

FIG. 2. RABBIT ANTIHUMANRED CELL ANTISERUM: OS-

MOTIC FRAGILITY OF ALBUMIN-PROTECTED AND CONTROL

CELLS AFTER INCUBATION. See Figure 1 for the compo-

nents of each incubation mixture and their proportions;

albumin was added in 2.6 mMconcentration.

100 7

614

DEXTRAN40 CONG. ADDEDTO MEDIUM(MILLIMOLAR)

FIG. 3. THE EFFECT OF DEXTRAN 40 ON LOSS OF PO-TASSIUM AND HEMOGLOBIN PRODUCEDBY RABBIT ANTI-HUMANRED CELL ANTISERUMAND C'. Each sample con-tained 0.2 ml red cells, 1.6 ml fresh serum, 2.4 ml Tris-NaCl buffer, and 0.12 ml antiserum.

present in concentrations twice the fully protec-tive concentrations of Dextran 40, Dextran 10still fails to offer protection against hemoglobinloss. Sucrose similarly failed to protect againsthemolysis. Consequently, both Dextran 10 andsucrose may be presumed to have smaller molec-ular dimensions than the defect in the membraneproduced by antibody and C'. Dextran 20, how-ever, had an effect intermediate between that ofDextran 10 and Dextran 40, as illustrated inFigure 5. Concentrations of Dextran 20 higherthan 4.5 mMappeared to decrease C' activity asevidenced by a decrease in loss of potassium, andtherefore it was not possible to study concentra-tions of Dextran 20 that would protect com-pletely against hemolysis. The concentration ofDextran 20 that decreased hemoglobin loss to50% of the nonprotected control value was, how-ever, approximately double the concentration ofDextran 40 that afforded similar protection. Thissuggests that approximately 50%o of the Dextran20 molecules are protective and 50%o nonprotec-tive, presumably because the latter are smallerthan the size of the defects in the cell membrane.

To test this interpretation, the protective effectof a 1: 1 mixture of Dextran 40 and Dextran 10was compared with that of Dextran 20 and is il-lustrated also in Figure 5. That the molar con-centrations of this Dextran 40-10 mixture thatwere protective were nearly identical with the

979

eu


100

80'

I.-60

40-

20

0

K LOSS

HMGOINLOSS

08 1 6 2.4 32 40

DEXTRAN10 CONC. ADDEDTO MEDIUM(MILLIMOLAR)

FIG. 4. THE EFFECT OF DEXTRAN 10 ON LOSS OF PO-TASSIUM AND HEMOGLOBIN PRODUCEDBY RABBIT ANTI-

HUMANRED CELL ANTISERUM AND C'. Each sample con-

tained 0.2 ml red cells, 1.6 ml fresh serum, 2.4 ml Tris-NaCl buffer, and 0.11 ml antiserum.

protective concentrations of Dextran 20 supportsthe impression that the Dextran 20 curve can beaccounted for by the behavior of the 50%o of theDextran 20 molecules that are protective, whereasby analogy to the Dextran 40-10 curve, the per-meating Dextran 20 molecules would not be ex-pected to alter the protective effort of the non-permeating molecules. Information about themolecular size distribution curve of these dex-trans, based on light scattering (Mw),9 indicatesthat approximately 98% of Dextran 10, which issmaller than the membrane defect, is composed ofmolecules less than 20,000 in molecular weight.Ninety per cent of Dextran 40, which is largerthan the defect, is greater than 20,000 in molecu-lar weight, and the median molecular weight ofDextran 20 is 20,000. Thus, the membrane de-fect has approximately the same dimensions asdextran having a molecular weight (Mw) of 20,-000 as measured by light scattering.

Studies of the rabbit antihuman red cell anti-serum carried out with types 0, AB, A, and Bcells all reveal a similar pattern of protection byDextran 20, Dextran 40, and albumin againsthemolysis but no protection against loss of po-tassium.

These studies with rabbit antihuman red cellserum indicate that its action, with C', on the

9 Kindly provided by Dr. Edward F. Grassl of Phar-macia Fine Chemicals, Inc., New York, N. Y.

cell membrane produces defects with smaller ef-fective radius than hemoglobin, albumin, Dextran40, and 50% of Dextran 20 molecules and largerthan Dextran 10, 50%o of Dextran 20, or sucrosemolecules; thus, red cell swelling must take placebefore actual hemolysis occurs. Hemolysis bythis antibody and C' proceeds by a colloid-osmoticmechanism. These results agree with those ofGreen and associates (13) in their studies of theeffects of rabbit hemolysin on mouse erythrocytes.Confirmation of the colloid-osmotic hemolyticeffect of this antibody was obtained by evaluationof the prehemolytic volume of unhemolyzed cellsseparated from hemolyzed cells after an unpro-tected incubation in which there was 57%o hemo-lysis. Although counting of the agglutinated cellswas not possible, estimation of the mean cell vol-ume (MCV) was made by calculation of the meancorpuscular hemoglobin concentrations (MCHC)before and after incubation. Final MCV= origi-nal cell volume x [MCHC (original)/MCHC(final)]. After a 90-minute incubation the con-trol cells with C' inactivated were found to havea volume of 85 3, and the damaged but unhemo-lyzed cells had a volume of 114 y3.

100

90

80

70

60CK

t012 50-

40-

30-

20-

10o

- K LOSS 0-0 HEMOGLOBINLOSS

DEXTRAN20

DEXTN 10

DEXTRAN20

DEXTRAN40+10

DEXTRAN40

O 2 3 4 5 6 7DEXTRANCONC. ADDEDTO MEDIUM

(MILLIMOLAR)

FIG. 5. COMPARISONOF THE PROTECTIVE EFFECTS OFDEXTRAN20 WITH DEXTRANS40 AND 10. Each sample con-tained 0.1 ml red cells, 0.8 ml fresh serum, 1.2 ml Tris-NaCl buffer. Only the loss of potassium in the Dex-tran 20 experiments is represented. The concentrationslabeled Dextran 40 + 10 represent the total molarity con-tributed by a 1: 1 mixture of the two dextrans.

980

MECHANISMOF IMMUNEHEMOLYSISANDANTIBODY SPECIFICITY

Studies utilizing human anti-A. The previousexperiments were repeated to compare the effectsof human anti-A and C' on group A1 human redcells with those obtained with rabbit antihumanred cell serum. Figure 6 shows the results ofsuch a study. Albumin failed to provide thecomplete protection against hemolysis observed inthe experiments with rabbit antihuman red cellantibody.

Since the bovine serum albumin molecule islarger than human hemoglobin, we concludedthat the action of human anti-A and C' producedred cell membrane defects large enough to permitthe direct escape of hemoglobin without the neces-sity of prior cell swelling. To support this con-clusion, an experiment was designed in whichhuman hemoglobin was added to the extracellularmedium. It was not technically possible toachieve as concentrated solutions of hemoglobinin the medium as had been utilized with albumin.Nevertheless, Figure 7 illustrates that at molarconcentrations of extracellular hemoglobin analo-gous to concentrations of albumin (Figure 1),which were significantly protective against anti-human red cell serum, hemoglobin failed to in-hibit hemolysis. Weconcluded that human anti-Aantiserum, in contrast to rabbit antihuman redcell antiserum, produced membrane defects ofsufficient size to permit hemoglobin escape fromthe cell directly without prior cell swelling. Theresults agree with and extend the observation ofJacob and Jandl (28) that addition of sucrose to

100'

80'

q.4J

60

40-

20

K+ LOSS

HEMOGLOBINLOSS

O 0.63 1.26 1.89 2.52 3.15

ALBUMIN CONC. ADDEDTO MEDIUM(MILLIMOLAR)

FIG. 6. THE EFFECT OF ALBUMIN ON LOSS OF POTAS-

SIUM AND HEMOGLOBIN PRODUCEDBY HUMANANTI-AAND C'. Each sample contained 0.1 ml red cells, 2.0 mlfresh serum, and 0.3 ml antiserum.

100-

80

k, 60 -

40-

20-

HEMOGLOBINLOSS

0 0.46 0.92 1.38 1 84

HEMOGLOBINCONC. ADDEDTO MEDIUM(MILLIMOLAR)

FIG. 7. THE EFFECT OF HEMOGLOBINON LOSS OF PO-

TASSIUM AND HEMOGLOBINPRODUCEDBY HUMANANTI-AAND C'. Each sample contained 0.1 ml red cells, 2.0 mlfresh serum, 2.0 ml hemoglobin solution, and 0.3 mlantiserum.

the medium did not prevent hemolysis by humananti-A and C'.

Studies utilizing fractionated antisera. Good-man (29) and Johnson, Woernley, and Pressman(30) have found that the rabbit produces 7 S Y2hemolysin when immunized repeatedly withhuman red cells. Hemolytic activity in humananti-A sera has been found in both 7 S and 19 Sfractions by McDuffie, Kabat, Allen, and Wil-liams (31). The 7 S fraction of the rabbit anti-human red cell serum was incubated with humangroup A1 red cells with and without added al-bumin; the results of this experiment are shownin Table I. Loss of hemoglobin produced by the7 S fraction of the rabbit antihuman red cellantibody was markedly inhibited by albumin,whereas loss of potassium was unaffected. Theseresults are identical to those observed with theunfractionated antiserum. The total loss of hemo-globin and potassium produced by the amount ofthe 7 S fraction of human anti-A employed inthese experiments did not exceed 28%; therewas still no significant inhibition of loss of hemo-globin relative to loss of potassium in albuminprotected tests. These results are comparable tothose obtained with the unfractionated anti-Aserum. In summary, the 7 S antibody fractionscontaining the hemolytic activity of these antiserabehaved like the respective whole native sera interms of the size of the defects induced in thered cell membrane. The size of the membrane

981


TABLE I

The 7 S fractions of rabbit antihuman red cell antiserum andhuman anti-A: the effect of albumin on loss of potassium

and hemoglobin

Albuminconcen-tration Potas- Hemo-

added to sium globinAntiserum medium loss loss

mM % aRabbit 0 68 58

antihumanred cell* 3.2 77 12

Human 0 28 26anti-At 3.2 22 13

* Each sample contained 0.2 ml red cells, 1.6 ml serum,and 0.2 ml antiserum fraction.

t Each sample contained 0.1 ml red cells, 2.0 ml serum,and 0.3 ml antiserum fraction.

defect is not apparently related primarily to themolecular species of antibody involved, in thattwo 7 S antibodies of comparable molecular size,but of different origin and specificity, producedmembrane defects of different sizes.

Studies utilizing rabbit antihuman A. Whenantihuman A of rabbit origin was incubated withhuman group Al red cells and C', albumin addedto the medium did not significantly inhibit cellularloss of either potassium or hemoglobin (TableII). Thus, rabbit antihuman A produced mem-brane defects of greater size than the hemoglobinmolecule. Its action in this respect was similarto human anti-A and different from rabbit anti-human red cell serum. The difference in resultsbetween the two antisera of rabbit origin, but ofdifferent specificity, indicates that the size of themembrane defect created by antibody and C' doesnot appear to be a characteristic of the animalspecies in which the antibody is produced.

TABLE II

Rabbit anti-A: the effects of albumin on lossof potassium and hemoglobin*

Albuminconcen-tration Potas- Hemo-

added to sium globinmedium loss loss

mM % A0 60 471.6 63 363.2 66 34

* Each sample contained 0.2 ml red cells, 3.0 ml serum,and 0.4 ml antiserum.

Rather, the characteristic size of the membranedefect appears to be related to the antigen againstwhich the antibody is directed, i.e., the immuno-logical specificity of the antigen-antibody reaction.

Discussion

In these experiments, size of the antibody-C'-induced membrane defects can be defined in termsof the size in solution of molecules which do ordo not protect the cells against colloid osmoticlysis. A variety of methods have been used toassess the size and dimensions of macromolecules,and results have varied widely depending on tech-niques employed and the physical state of themolecules under the conditions of the test, as hasbeen discussed by Edsall (32). Based on X-raystudies of human albumin, Low (33) has sug-gested that the molecule has an elongated shape.

TABLE III

Stokes-Einstein equation and values of effectivediffusion radius for various substances

RT

RES =TRs=6r,rDN*

Molecule Mol Wvt RES in A References

Sucrose 342 4.4 40, 41Dextran 6,000 16.8 43Dextran 16,000 28.3 43Dextran 18,000 30.1 43Dextran 20,000 32.0 43Hemoglobin 68,000 32.5 44Albumin 69,000 35.5 43, 44

RES = effective diffusion radius, R = gas constant, T =temperature, 77 = viscosity of water, D = diffusion co-efficient, and N = Avogadro's number.

A spherical model fits data obtained on bovinealbumin solutions by Loeb and Scheraga (34).Similarly, dextrans have been described as along slender molecule by Gronwall (35) and byIngelman and Halling (36); Ogston and Woods(37, 38) have suggested that more symmetricalmodels fit the data better. Hemoglobin, even inthe unhydrated state, has been described as afairly symmetrical molecule by Perutz (39).

To describe the diffusion behavior of largemolecules in solution through porous membranes,the effective diffusion radius, calculated fromthe Stokes-Einstein equation (Table III), hasbeen demonstrated by Pappenheimer (40), Pap-penheimer, Renkin, and Borrero (41), and Ren-

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kin (42) to provide a useful and valid estimate ofmolecular size. The effective diffusion radius,simply stated, is the radius of a sphere withequivalent diffusion coefficient. Values of effec-tive diffusion radii for the substances used in theseexperiments were taken from the literature andare listed in Table III.

By relating these estimates of molecular sizeto the present experimental results, the size of theantibody-C' induced membrane defects can bedefined more specifically. The holes created bythe rabbit antihuman red cell serum and humanC' are approximately equal to the median molecu-lar size of Dextran 20, molecular weight 20,000,or 32 A in effective radius. On the other hand,the holes produced by antihuman A of eitherhuman or rabbit origin and human C' must begreater than 32.5 A in effective radius. Theseestimates depend, of course, on the validity of theestimates of molecular size employed and theassumption that there are no significant inter-actions between the test molecules and the mem-brane "holes." They do, however, provide esti-mates of the relative size, in a functional sense, ofthe membrane lesions induced by these differentantibodies and human C'.

In his original description of colloid-osmotichemolysis, Wilbrandt considered lysis of sensi-tized sheep cells by C' to be nonosmotic on thebasis of the shape of osmotic fragility curves (9).However, as Ponder (45) has pointed out, thecritical volume for osmotic lysis in a variety ofhemolytic systems is very little in excess of theinitial volume. Different critical hemolytic vol-umes have been observed in the course of erythro-cyte lysis by different agents, and the colloid-osmotic theory does not elucidate the nature ofthis effect of lysins. Jacob and Jandl (28) havepresented evidence that sucrose inhibits hemolysiscaused by sulfhydryl inhibitors and that in thiscase lysis occurs by a colloid osmotic mechanism.These authors found that addition of sucrose tothe medium did not prevent hemolysis of group Ahuman red cells by human anti-A and C' andsuggested that relatively large membrane defectswere produced, a finding consistent with ourresults.

The maintenance of normal intracellular potas-sium levels in the erythrocyte depends upon thebalance between normal potassium leakage from

the cells in the direction of the electrochemicalgradient and active transport of potassium intocells against this gradient by energy-requiringprocesses. The question might be raised whetherthe red cell potassium loss we observed couldsimply be the result of inactivation of the "potas-sium pump" allowing the normal potassium leakto proceed unopposed, as, for example, Eckel(46) has shown in red cells incubated with smallconcentrations of sodium fluoride. This questionis answered by consideration of the normal rateof potassium leakage from red cells determined bySheppard and Martin (47) and Ponder (48, 49),which is far too slow to produce potassium lossesof the magnitude observed after 2-hour incuba-tions in the present studies.

The structural counterpart of the cell membraneof these physiologically demonstrated defects in-duced by antibody and C' is not known. Theyare much larger than the normal pores of thehuman red cell membrane, estimated by Solomon(50) to have a radius of about 3.5 A. The defectsin the membrane, at least in the case of the moreprecisely defined lesion induced by the rabbitantihuman red cell serum, is of the same generalorder of size as the molecular cross sectional areacalculated for rabbit antibodies against sheep cellsby Heidelberger, Weil, and Treffers (51). Latta(52), studying antibody-lysed sheep erythrocyteswith the electron microscope, described surface"cracks" in the affected cells. Goldberg andGreen (11) have observed focal pouching andfolding of the cell membrane with the electronmicroscope in ascites tumor cells treated withantibody and C'.

Complement alone does not appear to determinethe size of the induced red cell membrane de-fects. Human C' was utilized in all of our ex-periments, and yet the size of the membrane de-fects varied with different antisera. The hy-pothesis may be proposed that the antigenic sitesmay have been critical in determining the sizeof the defect created by antibody and C'. All ofthe specific antigen(s) recognized by the antibodyin the rabbit antihuman red cell serum used inthese experiments is not known. Clearly, how-ever, the antibodies were not directly primarilyagainst antigens of the human ABOsystem. Therabbits were immunized with human group 0red cells, and the antiserum, when tested against

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the erythrocytes of all the human ABO groups,produced similar membrane defects that weresmaller than those induced by anti-A sera.

Little is known of the distribution of antigensites or functionally active sites on the surface ofthe red cell membrane. The technique employedin these experiments may provide an approach bywhich to evaluate whether the size of the erythro-cyte membrane defect has a relationship to anystructural characteristic of the involved antigens.

Summary

1. Rabbit antihuman red cell serum and humancomplement (C') produce functional holes in hu-man erythrocyte membranes that are 32 A in ef-fective radius. Destruction of these injured cellsin vitro ensues by colloid-osmotic lysis.

2. In contrast, antihuman A of rabbit andhuman origin with human complement producesholes greater than 32.5 A in radius, permittingdirect escape of hemoglobin.

3. The size of the membrane defect is not re-lated either to molecular size of the antibodymolecules or to the species of animal producingthe antibody.

4. We suggest that specificity of the antigenicsites involved determines the size of the mem-brane defect produced. In turn, the mechanismof the complement-dependent hemolysis is deter-mined by the size of the induced membranedefects.

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differences in the mechanism in vitro immune hemolysis related...

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