[CANCER RESEARCH 38, 4534-4539, December 1978]0008-5472/78/0038-0000$02.00

Selective Reductionof Human Tumor Cell Populationsby HumanGranulocytesin Vitro1

Darwin 0. Chee,2 Courtney M. Townsend, Jr.,3 Mary Ann Galbraith, Frederick R. Ellber, and Donald L.MortonSurgicalService,VeteransAdministrationHospital,Sepulveda,California91343(0. 0. C., C. M. T.,M. A. G., F. A. E., D. L. M.J,and Divisionof Oncology,Department of Surgery, UCLA Schoolof Medicine, Los Angeles, California 90024(D. 0. C., C. M. T., F. A. E., D. L. M.j

ranged from normal cells, including normal embryonic andadult fibroblasts, to various histological types of tumor cells(Table 1). These cell lines were propagated in SCEM.4Duringthesestudieswe discoveredthatsome ofthesecelllines were contaminated with Mycop!asma hyorhinis. Subsequently, Mycop!asma-free cultures were exposed to granulocytes, and identical results were obtained, indicatingthat our results could not be explained by Mycoplasmainfection of the cell lines.

Preparationof Granubocytes.The procedurefor isolatinggranubocytes from human blood, as described by Boyum(2), was used with minor modifications. Twenty ml of bloodwere collected into a syringe containing 2 ml of 1000 p1heparin (Organon, Inc., West Orange, N. J.) and 2 ml of 6%dextran (Sigma Chemical Co. , St. Louis, Mo.). After themixture had been agitated, the syringe was inverted andplaced in a 37°incubator for 1 hr. The leukocyte-rich plasmawas collected in a 50-mb test tube by means of a Butterfly10 infusion set (Abbott Laboratories, North Chicago, III.)

and centrifuged at 2000 x g for 15 mm. The volume ofsupernatant plasma was reduced to 7 ml, and 14 ml Dulbecco's phosphate-buffered saline (Microbiological Associates)were added tothetube.The pelletofleukocyteswasdispersed, and the leukocyte suspension was carefullylayered on 28 ml Ficoll-Hypaque solution (1). The granulocytes were separated from the agranulocytes by centrifugation at 1460 rpm for 45 mm. The supernatant and interface layer containing the agranulocytes were siphoned offbefore the pellets of granubocytes were washed 2 times with30 ml phosphate-bufferedsaline.The granubocyteswereresuspended in 10 ml SCEM. Viable granubocytes weiecounted by the trypan blue exclusion method, and theirnumber was adjusted to 1 x 10°/mb.

In Vitro Test with Granulocytes. Target cells at 1 x 10@cells/mb in SCEM were seeded with 1 ml of the cell suspension in a tissue culture plate (Cluster 24; Costar, Cam

bridge, Mass.). The plate was placed on a rocker platform(Bellco Glass, Inc., Vineband, N. J.) in a moist CO., incubatorat37°for30 mm and thenatrestinthe incubatorforanadditional 4 hr. One ml of the granubocyte suspension wasthen added to appropriate cultures. Control wells received1 ml SCEM; the cocultures of target cells and granulocyteswere rocked in a 5% CO2humidified 37°chamber for 30 mmand then incubated without agitation for 48 hr. The cultureswere washed, fixed with 4% formaldehyde, stained with10% Giemsa stain, read by visual inspection, and scoredfrom —to4+.

4 The abbreviations used are: SCEM, Chess essential medium (Microbio

logical Associates, Bethesda, Md.), supplemented with 10% fetal calf serumandgentamicin(50/mg/mI);[‘25l]ldUrd,5-['@'I]iodo-2-deoxyuridine.

ABSTRACT

Humanperipheralbloodgranubocytesat a 10:1effectorceII:target cell ratio were shown by an in vitro semiquantftativestainingprocedureto reducethe numberof humantumor cells, but not that of normal cells. Microscopicobservations revealed that this selective reduction oftumorcellsbygranubocyteswas a functionof bothdetachment and cytolysis.The cytotoxiceffect of granubocyteson the tumor cells was confirmed by a quantitative 5-[‘25l]iodo-2-deoxyuridinerelease assay.The data indicatethat humangranulocytesat a relativelyloweffector:targetcell ratio(10:1)havethe capacityto recognizeanddestroyhumantumorcellsIn vitro.

INTRODUCTION

During studies to determine the role of macrophages inhuman tumor biology by an in vitro cytotoxicity assay, weconsistently observed that granubocytes from excoriatedskin significantly diminished the number of tumor cells, butnot that of normal cells.

Other investigators have reported that intact human granubocytes caused reduction of human fibroblasts by detachment of the target cells in a plaque assay (18) or at higheffector:target cell ratios (20). Destruction of tumor cells byintact granulocytes (3) or by the peroxidases of granubocytes was observed in murine systems (4, 6), and granubocytes were shown to kill antibody-coated target cells (13).Recently, it was demonstrated that circulating granubocytesfrom tumor-bearing mice specifically killed tumor cells invitro (7). In this report, we present evidence to show thathuman peripheralblood granulocytesat a relativelyloweffector:target cell ratio can selectively decrease populations of human tumor cells without diminishing the numberof normal human cells. The data also indicate that reductionof the tumor cells, not of normal cells, was attributable toboth detachment and cytotoxicity by the granubocytes.

MATERIALSAND METHODS

Target Cells. The target cells used for these studies

1 Supported by the Medical Research Service of the Veterans Administra

tion (Projects 1384, 2248, and 5854) and by USPHS Grants CA12582 andCA05262 awarded by the National Cancer Institute (Department of Health,EducationandWelfare).

2 Present address: Clinical Immunology, City of Hope National Medical

Center, Duarte, Calif. 91010.3 Recipient of NIH Fellowship 1-F22CA00843-O1 . Present address: Depart

ment of Surgery, U. S. Naval Medical Research Center, Bethesda, Md.20014.

Received February 27, 1978; accepted September 8, 1978.

4534 CANCER RESEARCH VOL. 38

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DesignationOriginSourceHEMWhole

embryoMicrobiologicalAssociatesMicro-2Embryonic

foreskinMicrobiologicalAssociatesUCLA-SO-MF14Adult

skinfibroblastUCLA@SOaUCLA-S0-SVA-MF23AdultskinfibrobbastUCLA@S0@SVAbUCLA-SO-SF,

UCLA-S0-M14UCLA-SO-SVA-M23Adult

skin fibroblastMelanomaMelanomaUCLA-SO°

UCLA-S0°UCLA@SO@SVAbMBA/9812Bronchogenic

carcinomaW.A. Nelson

Rees―HT1O8OOsteosarcomaW.A. Nelson

Rees―SK-MES-1Lungsquamouscell

carcinomaJ.Fogh―

Percentageof recoveryof granulocytesfrom 20 normal donorsbytheFicoll-HypaqueprocedureDonor

% granulocytesrecoveredaA.A.91S.A.87K.B.96MB.95T.B.93E.B.86M.D.86P.D.90W.F.93H.F.97MG.84C.G.97C.l.90D.P.83S.R.96A.S.89S.S.80D.T.94H.W.85G.Z.

88a

Mean ±SE. = 90.0 ±1.13.

Effect of Granu!ocytes on Tumor Ce!!s in Vitro

triplicate set of wells for minimum release, and triplicatesets of experimental wells, Dunnett's t test for one controland several treatments was used to analyze the data (17).

Quantitative effects of donor granubocytes on one normaland 2 tumor cell lines were compared by Student's t test.

RESULTS

Percentageof Recoveryof Granulocytesfrom Blood.Table 2 shows the percentage of recovery of granubocytesfrom 20 healthy normal donors with the use of a modifiedFicoll-Hypaque technique (2). The preparations contained80 to 97% granubocytes, with some mononuclear cells,mostly lymphocytes. The mean percentage of recoveredgranubocytes was 90.0 ±1.13% (S.E.). The granubocytepreparations usually contained a small number of erythrocytes which did not interfere with the test.

Method Used in Scoringthe Test Plate. Chart 1 illustrates a typical test plate and the scoring criteria used forthis assay system. Because there was no discernible difference in the stain intensity of the control and normal cells inthe 3 left columns, the experimental cells were scored asminus (—).In contrast, tumor cells in the 3 right columns ofwells showed varying staining intensity ranging from 1+ to4+ when compared to control wells. Initially, this semiquantitative method for comparing the effect of granubocytes onneoplastic cells versus that of normal cells was used.Subsequently, a more precise quantitative radioisotoperelease assay was used to demonstrate a cytocidal effect ofgranulocytes on tumor cells (see below).

Effectof VaryingRatiosof Granulocytesto Tumor andNormal Cells. The effect of varying ratios of effector totarget cells is shown in Table 3. At 5:1 ratios, only minimalor no discernible difference was seen between control andexperimental wells with either normal or neoplastic cells.By contrast, at effector cell:target cell ratios of 10:1 or 20:1,the granubocytes selectively reduced the population of tumor cells, but not that of normal cells. However, at a ratio

Table 2

Table 1Human normal and tumor cell lines used as target cells

a University of California, Los Angeles, Surgical Oncology.

b University of California, Los Angeles, Surgical Oncology, and

Sepulveda Veterans Administration Hospital.C Cell Culture Laboratory, Naval Biomedical Research Labo

ratory, Oakland,Calif. 94625.(I Sloan-Kettering Institute for Cancer Research, Donald S.

Walker Laboratory, 145Post Rd., Rye,N. Y. 10580.

Quantitative[125b]ldUrdReleaseAssay.Targetcellsthatefficiently incorporated [‘25b]IdUrd(Amersham/Searle Corp.,Arlington Heights, Ill.) were selected for the quantitativerelease assay. Target cells were prelabeled 24 hr in advancewith [1251]ldUrdin the ratio of 100 pCi [‘25I]bdUrdfor every 1x 106cells.The labeledcellswere harvestedandwashed3times with 50 ml SCEM . The cell number was adjusted to5.6 x 10°cells/mb, and 25 p1 of cell suspension were seededrandomly into wells of a Falcon Microtest II plate (FalconPlastics, Oxnard, Calif.). This number of cells was chosento approximate comparable confluency, as in wells of theCostar cluster plates, under the assumption that closecontact between effector and target cells is essential tobring about an effect on the target cells. The microtest platewas incubated in a humidified CO2 incubator at 37°for 4 hrbefore 0.1 ml of granulocyte suspension containing 1.4 x10°cells/mb in SCEM (a 10:1 effector:target cell ratio) wasadded to test wells. The assay was done in triplicate,including controls for maximum and minimum release of[125blIdUrd from the target cells. Maximum release wasachieved by adding 0.1 ml of 10% NaOH to triplicate targetcell cultures; minimum release was obtained by adding 0.1ml SCEM to triplicatetargetcellcultures.The platewasgently rocked in a humidified CO2 incubator at 37°for 30mm and was then stationary for 48 hr. Supernatants fromall wells were collected by the Titertek supernatant collection system (Flow Laboratories, Rockville, Md.). In thissystem supernatants are collected from wells of a FalconMicrotest II plate by cylindrical wicks without disturbing thecells in the wells. The wicks containing the adsorbedradioactivity were placed in a ‘ycounter. The percentage ofcytotoxicity was computed from the mean triplicate countswith the use of the following formula:

. . test cpm— minimum cpm% cytotoxlclty . x 100maximumcpm

Statistical Analysis. Since each microtest plate contamed a triplicate set of wells for maximum release, a

DECEMBER 1978 4535

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NormalcelllinesTumorcelllines•UCLA

VAHS/SOMBA/RatiosHEMMicro-21SFM149812HT1O8O320:14+4+4+4+4+4+160:14+3+3+4+4+3+80:13+1+2+4+4+3+40:12+—±4+4+3+20:1±—±4+4+2+10:1———4+3+2+5:1———2+±—2.5:1———1+——1.25:1———1+——

Normal cell strainsTumor celllinesUCLA

UCLA SOMBA/DonorHEMMicro-2SO-FlM149812HT1O8OP.B.———2+2+1+M.B.--—2+2+1+C.F.———3+2+1+B.H.—-—2+2+±J.J.———2+2+1+M.K.———2+2-i-2+J.P.±——4+2+1+D.P.±——3+3-'-4+R.P.±——4+3+2+L.S.———3+2+1+D.T.———3+3-'-1+W.T.———4+4+1+

0. 0. Cheeetal.

the microscopic observations and to quantitate the amountof cytotoxicity of one normal and 2 tumor cell lines onexposure to granulocytes from 6 donors. The data (Table 6)showed that none of the 6 donors' granubocytes displayedsignificant cytotoxicity to the UCLA-SO-SF fibroblasts (p >0.05). By contrast, the granubocytes from the 6 donors weresignificantly cytotoxic to both the HT1O8Osarcoma and theSK-MES-1 lung carcinoma (p < 0.005). Furthermore, thegranubocytes significantly killed the HT1O8Oosteosarcomacells more than the SK-MES-1 lung carcinoma cells (p <0.0005).

DISCUSSION

The data obtained from this investigation indicate thathuman peripheral blood granubocytes preferentially diminished populations of human tumor cells, but not those ofhuman normal cells, in vitro when the granulocyte:targetcell ratio was 10:1 . The data further indicate that reductionof the number of tumor cells by granubocytes was mediatedby both detachment and cytolysis.

This recognition of the malignant state by the granulocytes appeared to be a general and naturally occurringphenomenon. The granulocytes of all 12 normal donorstested at a 10:1 effector:target cell ratio decreased thenumber of tumor cells, but not that of normal cells (Table

Table 3

HEMMICRO-UCLA-UCLA-MBA-HT2 SO-SVA80-4*149812 1080

SFo

NORMALCELLS TUMORCELLSChart 1. Effect of granulocytes on normal and tumor cells. Criteria used

for scoring plates. Top row of wells are control cultures without granulecytes; the 3 remaining rows are experimental cultures exposed to granulecytes from 3 donors.

of 40:1 there was indiscriminate reduction of target cells.This experiment was repeated twice, and the results inTable 3 are the mean scores of the 2 experiments. Subsequently, the 10:1 effector:target cell ratio was used in allother experiments.

Effectof Several Donors'Granubocyteson NormalandTumorCells.Table4 showstheeffectof granulocytesfrom12 normal donors on an arbitrary panel of 3 normal and 3

tumor cell lines. At the 10:1 ratio, granubocytes decreasedthe number of tumor cells, but not the number of normalcells. The degree of noxious effect of the 12 donors'granulocyteson the3 tumor celllinesvariedwiththedonorand thecellline.

Effect of Granulocyteson Paired AutologousNormaland Tumor Cells. Histocompatibility differences betweeneffector cells and target cells could account for the selectivereduction of tumor cells when it is assumed that the HLA ofthe donor granubocytes matched those of tumor cells, butnot those of normal cells (Table 4). Although such matchingis unlikely, this possibility was conclusively ruled out bycomparing the effect of granubocytes on 2 pairs of normaland corresponding autologous tumor cells. As shown inTable 5, the granubocytes of 5 donors significantly diminished the population of tumor cells, but not that of normalcells.

Quantitative Effect of Granulocyteson Tumor Cells.Microscopic observations revealed that besides their capability to detach tumor cells, the granubocytes were cytotoxicfor tumor cells, but not for normal cells. However, thedegree of cytotoxicity appeared to vary both with the donorgranubocytes and the cell lines. Because of this variability,a quantitative [125IJIdUrdrelease assay was used to confirm

CELLCONTROL

MM

CG

MK

Effect of varying ratios of granulocytes to target cells on normaland tumor cell lines

Table4Effect of granulocyteson norma!and tumor cells at a 10:1

granulocyte:targetcell ratio

4536 CANCERRESEARCHVOL. 38

CONTROL

DMM@ -

NCG

@ M K

D0N0R

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DonorCell line%cytotox

icity―p@)K.

B.C.F.J.J.KM.R.R.S.S.UCLA-SO-SF,18

120

131111NSC

NSNSNSNS

NSKB.

CF.J.J.KM.R.R.5.5.HT1O8O53

5339475460S

SSSS

SKB.

C.F.J.J.K.M.R.R.S.S.SK-MES-135

2327333530S

SSSSS

Effect of granulocytes on 2 pairs of normal and corrautologous tumor celllinesespondingDonorUCLA-SO-

UCLA-SO- UCLA-SOMF14 M14 SVA-MF23

(normal) (tumor) (normal)UCLA-SOSVA-M23

(tumor)G.P.

A.B.M.G.W.H.P.H.-

3+ —

- 3+ —

— 3+ —

— 3+ —— 3+ —3+

2+3+2+2+

Effect of Granulocytes on Tumor Cells in Vitro

4). However, a larger number of donor granubocytes mustbe tested in order fully to confirm our hypothesis. The 10%contamination of mononuclear cells in the granubocytepreparation was an unlikely explanation for the injuriouseffect to tumor cells, since most in Vitro lymphocytotoxicitytests require effector:target cell ratios of at least 100:1 (14,21).

Since activated macrophages have been shown to bedetrimental to tumor cells in Vitro (9, 10), it is conceivablethat the small percentage of macrophages that contaminated the granubocyte preparations might have accountedfor the observed cytotoxicity. However, the method used inseparating granubocytes from peripheral blood yielded, onthe average, 90% granubocytes and 10% mononuclear cells(Table 2). In such granubocyte preparations, approximatelyonly 1% monocytes (macrophages) are present in themononuclear cell population (2). Therefore, in the assayused in these studies the macrophage:target cell ratio was0.01 :1, a ratio that was inadequate for cytotoxicity orcytostasis by macrophages (9, 10). Furthermore, sincenormal healthy donors were used in this investigation, it isdoubtful that the macrophages of all these donors wereactivated. In addition, no known agent that activates macrophages was added to our in vitro assay system. Thus, it isimprobable that the cytotoxicity could be due to the macrophages that contaminated the granubocyte preparations.

HLA differences were ruled out as an explanation for thedifferential effect, because choice of donor granubocyteswas random and a chance HLA match of normal cells, butnot one of tumor cells, was improbable. Furthermore, wecould findno evidencein the literatureforgranulocyterecognition of HLA differences. This conclusion was furthersubstantiated when the effect of granulocytes was compared on 2 pairs of autobogous normal and tumor cell linesin which both the fibroblasts and corresponding tumor cellswere derived from the same cancer patient. The granulocytes of 5 donors significantly reduced the 2 populations ofmelanoma cells, but not the 2 corresponding autologousnormal fibroblast cultures (Table 5). Therefore, selectivereduction of tumor cells by granubocytes is inconsistentwith the view of HLA histoincompatibility between effectorcellsand targetcells.

These data also are incongruent with the notion that thegranulocytes discriminated normal from tumor cells on thebasis of embryonic or fetal antigens present on tumor cells.Cell strains HEM and Micro-2 were embryonic cells, butneither was adversely affected by the granubocytes (Chart 1;Table 3).

Since both the tumor and normal cells used in thisinvestigation were propagated in medium supplemented

Table5

Table 6Quantitative cytotoxic effect of granulocytes on normal and tumor

cell lines

a Cytotoxicity of the 6 donors' granulocytes on UCLA-SO-SF,:

compared to HT1O8O,p < 0.0005, compared to SK-MES-1, p <0.0005. Cytotoxicity of 6 donors' granulocytes on HT1O8Ocomparedto SK-MES-1,p < 0.0005.

b Statistical analysis was done by comparing triplicate cultureswith and without granulocytes with the use of Dunnett's t test.

C NS, not significant; S, significant.

with fetal calf serum, the heterologous membrane antigen(1 5) was excluded as the reason for granulocyte activity

against tumor cells. Likewise, in vitro antigens induced byin vitro culture conditions (16) were ruled out as an explanation for the selective action of granulocytes on tumorcells. Both tumor and normal cell lines were propagated invitro for at least 6 weeks before these experiments wereconducted, and all should have contained the antigensinduced by tissue culture environment.

Microscopic examinations showed that the reduction ofadherent tumor cells by granulocytes was brought about bydetachment and cytolysis (Fig. 1). At the ratio used, thecytolytic effect was not always obvious. However, our datawith the [125l]ldUrd release assay confirmed the microscopicobservations (Table 6). In contrast to these observations, a°1Crrelease assay showed only detachment without anytumor cell lysis (20). The reasons for this discrepancy areunknown. There are several possible explanations. Theseinclude differences@n isotopes used in labeling target cells,assay systems, cetl lines, incubation time, and culturevessels. A combination of these factors was probably responsible for the discordance between our data and thoseofothers.

Our data with the quantitative [‘25l]bdUrdrelease assayfurther suggest that granulocytes are more cytocidal forsarcomas than for carcinomas. The granubocytes of all 6donors tested were more cytotoxic at a statistically significant level (p < 0.0005) to the HT1O8Oosteosarcoma cellsthan to the SK-MES-1 lung carcinoma cells (Table 6). Wededuce, therefore, that granulocytes are more destructiveto tumors that are derived from the mesoderm than totumors that originated from the ectoderm or endoderm.

4537DECEMBER 1978

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D. 0. Cheeet a!.

Alternatively, the HT1O8Ocells might have been more susceptible to the action of granubocytes than were the SKMES-1 cells, regardless of histological type or degree of

cancer (11). Most natural tumors in man are carcinomas,which suggests a possible phybogenetic relationship between the evolution of the granubocyte system and the

incidence of carcinomas. We do not know whether thisphenomenon has an in vivo correlate. However, evidenceexists that granulocytes function as a homeostatic surveillance system against neoplasia. In a rat system, tumor cellswere destroyed by neutrophilsin vitro,which correlatedwith a reduction of tumor transplants when the rats werestimulated to produce a large neutrophil response (4, 19).An intriguing question then is, ‘How do nascent neoplasmssurrounded by an inimical environment evade the complexand efficient reticuboendothelial system?―

Compelling evidence (8, 12) suggests that neutrophils(i.e., granubocytes)play a minor role in hyperacutealbograftrejection . Our data support this argument. Granubocyteswere relativelyinnocuousto normalalbogeneiccells(Chart1 ; Tables 4 and 5). Similar results were reported by another

group of investigators with one strain of normal fibroblasts(20). If these observations are valid, it just might be that the

rejection of an albograft and the resolution of a tumorinvolve different mechanisms. The former requires the immune system, whereas the latter necessitates a compendium of the lymphoreticular system, including the inflammatory response.

We do not propose that granulocytes are harmless tonormal cells. Our findings show unequivocally that at a 20:1effector:target cell ratio, granubocytes are injurious to normal cells (Table 3). Other investigators have also demonstratedthatgranubocytesare injuriousto normal cells(18,20). Moreover, others have observed the destructive effectof polymorphonuclear leukocytes in a hyperacute xenograftrejection model (22). There is evidence to indicate thatneutrophils destroy normal cells in such lesions as theArthus phenomenon, acute nephrotoxic nephritis, and thenecrotizing arteritis of acute serum sickness (5).

We submit, however, that during tumor rejection granubocytesare attracted at the site of the tumor bed and destroyboth tumor and normal cells but destroy relatively moretumor cells than normal cells. If the tumor is completelydestroyed, the natural process of growth, differentiation,regeneration, and repair ensue and result in areas of healednormal tissue.

4538 CANCER RESEARCH VOL. 38

REFERENCES1. Boyum, A. Isolation of Mononuclear Cells and Granulocytes from Human

Blood. Isolation of Mononuclear Cells by One Centrifugation, and ofGranulocytes by Combining Centrifugation and Sedimentation at 1 g.Scand. J. Clin. Lab. Invest., 21 (Suppl. 97): 77-89, 1968.

2. Boyum, A. Isolation of Lymphocytes, Granulocytes and Macrophages.5cand. J. Immunol., 5 (Suppl. 5): 9-15, 1976.

3. Clark, A. A., and Klebanoff, S. J. Neutrophil-Mediated Tumor CellCytotoxicity: Role of the Peroxidase System. J. Exptl. Med., 141: 1442-1447, 1975.

4. Clark, A. A., Klebanoff, S. J., Einstein, A. B., and Fefer, A. @roxidase1•120,Halide System: Cytotoxic Effect on Mammalian Tumor Cells. Blood,45: 161-170, 1975.

5. Cochran, C. G. Immunologic Tissue Injury Mediated by NeutrophilicLeukocytes.Advan.Immunol.,9:97-162.1969.

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7. Fisher, B., and Saffer, E. A. Tumor Cell Cytotoxicity by Granulocytesfrom Peripheral Blood of Tumor-Bearing Mice. J. NatI. Cancer Inst., 60:687-691,1978.

8. Forbes, A. D. C., Kuramochi, T., Guftman, A. D., Klassen, J., andKnaack, J. A Controlled Sequential Morphologic Study of HyperacuteCardiac Allograft Rejection in the Rat. Lab. Invest.. 33: 280-289, 1975.

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12. Knaack, J. Nonessential Role of Neutrophils as Mediators in HyperacuteRejection. Federation Proc., 35: 649, 1976.

13. Gale, A. P., and Zighelboim, J. Polymorphonuclear Leukocytes inAntibody-Dependent Cellular Cytotoxicity. J. Immunol., 114: 1047-10@1.1975.

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15. Inc. A. F., Inc. K., and Morton, D. L. Natural Antibody in Human Serumto a Neoantlgen in Human Cultured Cells Grown in Fetal Bovine Serum.J. NatI. Cancer Inst., 52: 1051-1058, 1974.

16. Irie, A. F., and Morton, D. L. A New Membrane Antigen on HumanCultured Cells. Proc. Am. Assoc. Cancer Res., 16: 171, 1975.

17. Li, C. C. Introduction to Experimental Statistics, pp. 419-423. New York:McGraw-Hill Book Co., 1964.

18. Lundgren, G., Zukoski, C. F., and Moller, G. Differential Effects ofHuman Granulocytes and Lymphocytes on Human Fibroblasts in Vivo.Clin. Exptl. Immunol., 3: 317-336, 1968.

19. Pickaver, A. H., Ratcliff, N. A., Williams, A. E., and Smith, H. CytotoxicEffects of Poritoneal Neutrophils on a Syngeneic Rat Tumor. Nature NewBiol., 235: 186-187, 1972.

20. Takasugi, M., Akira, D. , and Kinoshita, K. Granulocytes as Effectors inCell-Mediated Cytotoxicity of Adherent Target Cells. Cancer Res., 35:2169-2176, 1975.

21. Takasugi, M., Mickey, M. R., and Terasaki, P. I. Studies on Specificitiesof Cell-Mediated Immunity to Human Tumors. J. NatI. Cancer Inst., 537:1527-1538, 1974.

22. Winn, H. J., Baldamus, C. A., Jooste, S. V., and Russell, P. 5. AcuteDestructionby HumoralAntibodyof RatSkin Graftedto Mice.J. Exptl.Med., 137: 893-910, 1973.

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Effect of Granu!ocytes on Tumor Ce!Is in Vitro

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DECEMBER 1978 4539

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1978;38:4534-4539. Cancer Res   Darwin O. Chee, Courtney M. Townsend, Jr., Mary Ann Galbraith, et al.  

in VitroHuman Granulocytes Selective Reduction of Human Tumor Cell Populations by

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