induction of tumoricidal activity of …...rides tested, a linear /3-1,3-glucan structure and a...

[CANCER RESEARCH 45,1496-1501, April 1985]

Induction of Tumoricidal Activity of Polymorphonuclear Leukocytes by a Linear0-1,3-D-Glucan and Other Immunomodulators in Murine Cells1

Kaoru Morikawa,2 Reiko Takeda, Masatoshi Yamazaki, and Den'ichi Mizuno

Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Tsukui-gun, Kanagawa 199-01 [K. M., R. T., M. Y., D. M.], and Research Development Corporation ofJapan, Nissho-Building, 14-24, Koishikawa 4-chome, Bunkyo-ku. Tokyo 112[D. M.], Japan

ABSTRACT

The cytotoxic activity of polymorphonuclear leukocytes (PMN)against tumor cells induced in vitro by antitumor immunomodu-lators was examined by a 51Crrelease cytotoxicity assay. Among28 immunomodulators and other agents thus far tested, only ÃŸ-1,3-glucan from Alcaligenes faecalis var. myxogenes IFO 13140,Bacillus Calmette-GuÃ©rin,Propionibacterium acnes, zymosan A,

and Nocardia cell wall skeleton were found to cause induction.The cytotoxic activity of PMN with the /3-1,3-glucan was very

high, almost 100% cytolysis being observed at an effectortargetratio as low as 3. The other four potent immunomodulators hadeffects very similar to that of the 0-1,3-glucan. All five tumor celllines tested, MM46, MM48, MH134, EL-4, and YAC-1, were

lysed, whereas normal spleen and thymus cells and PMN werenot. Of four types of effector cells tested, PMN and casein-

induced macrophages were effective, whereas resident macrophages and J774 cells were not effective. The cytotoxic activityof PMN was greater than that of induced macrophages, althoughboth were induced by casein. From results on the polysaccha-rides tested, a linear /3-1,3-glucan structure and a minimumnumber average degree of polymerization of 125 of the 0-1,3-

glucan seemed to be required for induction of cytotoxicity ofPMN. The cytotoxic features of PMN and possible chemicalstructures of antitumor polysaccharides for induction of cytotoxicity are discussed.

INTRODUCTION

Previously (17), we reported early responses of cells in theperitoneal cavities of mice to antitumor immunomodulators, especially with respect to PMN3-inducing activity. Like bacteria,

many immunomodulators induced more PMN than did conventional inducers. However, we did not determine whether PMNact as effector cells against tumors in cooperation with immunomodulators.

In this work, to determine the in vitro PMN response toantitumor immunomodulators as foreign substances, we studiedthe cytotoxicity of PMN against tumor cells in vitro in the presence of immunomodulators. We have reported the effector activities of PMN in antibody-dependent and lectin-dependent cell-

mediated tumor cell cytotoxicities in vitro (28, 29), and there are

1This work was supported by a Grant-in-AkJ for Scientific Research from the

Ministry of Education, Science and Culture of Japan.' To whom requests for reprints should be addressed.* The abbreviations used are: PMN, polymorphonuclear leukocytes; TAK, 0-1,3-

glucan from Alcaligenes taecalis var. myxogenes IFO 13140; E:(T) ratio, effectortarget ratio; DPN, number-average degrees of polymerization; PCS, fetal calfserum; BCG, Bacillus Calmette-GuÃ©rin;Nocardia CWS, Nocardia cell wall skeleton;CM-TAK, carboxymethyl-/3-1,3-glucan; PMA, phorbol myristate acetate; RPMI,Roswell Park Memorial Institute Tissue Culture Medium 1640.

Received 8/10/84; revised 11/19/84; accepted 12/27/84.

several reports indicating that PMN can be effector cells in tumorcell cytotoxicity and tumor rejection (5, 8, 15, 18, 22). Manyimmunomodulators have been developed as immunotherapeuticagents against tumors, and their mechanisms of action havebeen investigated extensively (1, 4, 7). However, there havebeen few studies on whether PMN mediate the antitumor activities of immunomodulators. In this work, we found that someimmunomodulators induce extremely potent PMN cytotoxicityagainst various tumor cells. One of these effective immunomodulators, TAK, is a chemically defined neutral polysaccharide.The possible reactive chemical structure of this polysaccharideis discussed in comparison with the structures of the polysaccharides thus far tested.

MATERIALS AND METHODS

Mice. Inbred male C3H/He and C57BL/6 mice were purchased fromCharles River, Japan (Kanagawa, Japan) and Shizuoka ExperimentalAnimal Farm (Shizuoka, Japan), and were used at 8 to 10 weeks of age.

Tumor Cells. In most experiments, MM46, a transplantable ascitestumor from a spontaneous mammary carcinoma, was inoculated intoC3H/He mice. MM48 mammary carcinoma and MH-134 hepatoma ofC3H/He mice and EL-4 lymphoma of C57BL/6 mice were maintained in

the ascitic form by weekly passage in the peritoneal cavity of syngeneicmice. YAC-1, a Moloney virus-induced lymphoma of A/Sn mice, was

maintained in suspension culture in RPMI (Nissui Seiyaku Co., Tokyo,Japan) with 10% PCS (Flow Laboratories, North Ryde, Australia). Thesetumor cells were used as targets of effector cells.

PMN. A volume of 2 ml of 8% sodium casein in 0.9% NaCI solution(saline) was injected into the peritoneal cavity of C3H/He mice, and 6 hlater the peritoneal exÃºdate was harvested in RPMI supplemented withpenicillin (100 units/ml; Banyu Pharmaceutical Co., Tokyo, Japan) andstreptomycin (100 ng/m\\ Meiji Seika Co., Tokyo, Japan). The peritonealcells (about 95% PMN) were harvested by centrifugaron at 300 x g for5 min and washed 3 times by resuspension in RPMI. Finally, they weresuspended in RPMI containing 5% heat-inactivated FCS.

Preparation of Other Effector Cells. Casein-induced macrophages

were obtained by the same procedure as were PMN except that theperitoneal exÃºdate was harvested after 5 days. The PMN contaminationin this fraction was less than 2%. Resident peritoneal cells were collectedwithout previous injection of a stimulant. J774 cells (a transformed,rnacrophage-like cell line) were maintained in suspension culture in RPMI

with 10% FCS. These cells were washed 3 times with RPMI before usedas effector cells.

Examination of Cell Populations. Peritoneal cells were suspended inRPMI containing 10% FCS, cytocentrifuged, and stained with Giemsastain (Merck, Darmstadt, West Germany). Then, the proportions of PMN,macrophages, and lymphocytes were determined.

Cytolytic Assay. Cytolysis was assayed as described previously (31).Briefly, MM46 tumor cells (2 x 106 cells/ml) were labeled with Na251CrO4

(100 MCi/ml) in RPMI with 10% heat-inactivated FCS for 1.5 h and then

washed 4 times. Peritoneal cells obtained 6 h after injection of 2 ml of8% casein solution into mice and target cells (usually 5 x 103 cells) with

or without an immunomodulator were mixed in wells (7 mm diameter) of

CANCER RESEARCH VOL. 45 APRIL 1985

1496

Research. on August 16, 2020. © 1985 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

PMN TUMORICIDAL ACTIVITY INDUCED BY IMMUNOMODULATORS

flat-bottomed microplates (Nunc, OK-4000 Roskilde, Denmark). Themixtures were incubated in 0.2 ml RPMI with 5% heat-inactivated PCSfor 16 h at 37 Â°Cunder 5% CO2 in air. Then, the plates were centrifugea

at 450 x g for 10 min, and the radioactivity of the supernatant wasmeasured. Cytolysis was calculated as

100 â€¢

% of cytolysis =Experimental count - control count

Maximum releasable count - control count x 100

The maximum release of 51Cr, measured as that obtained by freeze-

thawing labeled tumor cells 5 times, amounted to 70 to 80% of the totalcell-associated radioactivity. The control count, measured as the radioactivity released from labeled cells in the presence of an immunomodu-

lator without PMN, amounted to about 25% of the total radioactivity andwas similar to the count released from tumors spontaneously withoutPMN or immunomodulators. Values are given as averages for duplicatedeterminations.

Reagents. TAK, lower-molecular-weight TAK derivatives (25), andCM-TAK were kindly provided by Takeda Chemical Industries, Ltd.

(Osaka, Japan). Propionibacterium acnes was purchased from RIBIImmunochemical Research, Inc. (Hamilton, MA). Lentinan was kindlyprovided by Dr. G. Chihara, National Cancer Research Institute (Tokyo,Japan) and from Ajinomoto Co. (Kanagawa, Japan). Zymosan A, N-formyl-L-methionyl-L-leucyl-L-phenylalanine, <- and X-carrageenan, poly-inosinic-polycytidylic acid, and polystyrene latex beads (average diame

ter, 0.3 urn) were purchased from Sigma Chemical Co. (St. Louis, MO).A preparation of Nocardia CWS was kindly provided by Fujisawa Chemical Industries, Ltd. (Osaka, Japan). OK432, a preparation of Streptococcus haemolyticus, was a gift from Chugai Seiyaku Co. (Tokyo, Japan).Concanavalin A and wheat germ agglutinin were from E. Y. Laboratory(San Mateo, CA). Muramyl dipeptide and TC 13, a polysaccharide froma rare actinomycetes, Microellobosporia grÃsea,were gifts from DaiichiSeiyaku Co., Ltd. (Tokyo, Japan). Yeast mannan was a gift from SuntoryCo. (Osaka, Japan). Bestatin was purchased from the Protein ResearchFoundation (Osaka, Japan). Schizophyllan, a glucan from the culturefiltrate of Schizophyllum commune, was a gift from Kaken Seiyaku Co.,Ltd. (Tokyo, Japan). Sodium casein from milk was a product of TokyoKasei Kogyo, Ltd. (Tokyo, Japan). Glycogen and carbon were from WakoPure Chemical Industries (Osaka, Japan). NIH thioglycolate broth andlipopolysaccharide from Escherichia coli 0127:B8, were purchased fromDifco Laboratories, (Detroit, Ml). Dextran was a product of NakaraiChemicals, Ltd. (Kyoto, Japan). Levamisole was a gift from Kyowa HakkoCo. (Kanagawa, Japan).

Bacteria. BCG was obtained from Japan BCG Co. (Tokyo, Japan). Ecoli B, wild type, was cultured for 6 h in heart infusion broth (EikenChemical Co., Tokyo, Japan) and then washed 4 times with saline toremove the culture medium. Heat-killed E coli was prepared by heatingthe cells at 100 Â°Cfor 30 min.

Antitumor Antibody. The immunization procedure used was as described previously (31). Briefly, C3H/He mice resistant to syngeneicMM46 tumor were obtained by injection of tumor cells attenuated withmitomycin C (Kyowa Hakko Co.) and then 4 challenges with fresh tumorcells. The IgG fraction of immune serum of resistant mice was purifiedby gel filtration and then DEAE ion-exchange chromatography. The IgGfraction has antitumor activity in antibody-dependent macrophage-me-

diated cytotoxicity (32).

RESULTS

PMN Cytotoxicity in the Presence of Antitumor Polysaccharide TAK. PMN cytotoxicity induced with immunomodulatorswas examined in an assay system composed of PMN, theimmunomodulator, and MM46 tumor cells as a target. PMN inthe presence of TAK were capable of destroying tumor celltargets (Chart 1). The cytolytic activity of PMN was very high.

o4-1

o

M

-20

O 0.8 3 12 50 200Effector-to-target ratio

Chart 1. PMN cytotoxicity in the presence of TAK. PMN and 5'Cr-labeled MM46tumor cells were incubated at 37 Â°Cfor 16 h Â¡nmedium only (O) or in medium with

TAK at 10 M9/ml (A) or 100 ^g/ml (â€¢).After incubation, the radioactivity of thesupernatant was determined. Values are means for duplicate determinations.

TAK (pg/ml)

Chart 2. Dose-response curves of the effect of TAK on PMN cytotoxicity. PMNand "Cr-labeled MM46 tumor cells were incubated at 37 Â°Cfor 16 h in the presence

of various concentrations of TAK. The tumor cells were incubated at E:T ratios of3:1 (A) or 12:1 (â€¢)or in the absence of PMN (O). After incubation, the radioactivityof the supernatant was determined. Values are means for duplicate determinations.

and cytolysis was almost 100% at E:T ratios of 3 to 50 in thepresence of TAK (10 and 100 tig/m\) but less at the higher E:Tratio of 200. Neither TAK nor PMN alone showed significantcytotoxicity. Thus, the cytolytic activity of PMN in the presenceof the polysaccharide immunomodulator TAK was extremelypotent and depended on the E:T ratio.

Dose Dependency and Time Course of PMN Cytotoxicitywith TAK. The dose dependency of the effect of TAK on PMNcytotoxicity is shown in Chart 2. The dose-response curves with

TAK were similar at E:T ratios of 3 and 12, increasing abruptlyto almost 100% cytolysis with TAK at 6.3 ng/m\. TAK alone hadno direct cytotoxicity against MM46 tumor cells, as shown at anE:T ratio of 0.

The kinetics of PMN cytotoxicity with TAK was also examined(Chart 3). Culture supematants were assayed for release of 51Cr

at the indicated times after the addition of labeled MM46 targetcells. PMN cytotoxicity with TAK increased rapidly between 4


1497




TOO

80

i/i>>o4->>>

60

40

20

04 8 12 16Incubation time (hr)

Chart3. Time course of PMN cytotoxicity induced by TAK. PMN and 51Cr-

labeled MM46 tumor cells were incubated at an E:T ratio of 12:1 with TAK (100fig/ml) (â€¢)or without TAK (O) at 37 Â°C.At the times indicated, the radioactivity of

the supernatant was determined. Values are means for duplicate determinations.

0 0.8 12 50 200

Effector-to-target ratio

Chart 4. PMN cytotoxicity in the presence of P. acnes and lentinan. PMN and"Cr-labeled MM46 tumor cells were incubated at 37 Â°Cfor 16 h in the presence

of P. acnes (â€¢)or lentinan P) at 100 Â«g/mlor in medium only (O). Values aremeans for duplicate determinations.

and 7 h to the nearly maximal release. These results indicatethat the cytotoxic action of PMN with TAK is very strong andrapid with a lag period.

PMN Cytotoxicity in the Presence of Other Immunomodu-lators. We examined27 other immunomodulatorsand agentsfor their ability to induce PMN cytotoxicity. These compoundswere tested at doses of 0.1 to 100 ng/rr\\ with E:T ratios of 0.8to 200. Chart 4 shows the results with P. acnes and lentinan at100 Â¿Â¿g/ml,indicating that P. acnes was as effective as was TAKbut that lentinan did not induce PMN cytotoxicity. Table 1 summarizes the abilities of various immunomodulators to elicit PMNcytotoxicity. Like TAK and P. acnes, BCG, zymosan A, andNocardia CWS induced PMN cytotoxicity, but other immunomodulators and agents did not. Thus, the compounds testedwere either strongly effective or completely ineffective. The 5effective immunomodulators gave almost identical PMN cytotox-icities, inducing potent cytolytic activity with almost 100% cy-

tolysis at E:T ratios from 3 to 50 but less cytolytic activity at anE:T ratio of 200 (data not shown).

Target Cell Specificity. The specificityof this PMN cytotoxicity was examined with various kinds of 51Cr-labeledtarget cells

in the presence of TAK at 100 nQlm\ (Table 2). Two othersyngeneic tumor cell lines of C3H/He mice, MM48 and MH134,and other murine tumor cell lines, EL-4 and YAC-1, which areallogeneic to C3H/He mice, were lysed by PMN. These cytolyticactivities against all the tumor cells tested were as potent asthat against MM46 tumor cells. On the other hand, when PMNwere incubated with 51Cr-labeledspleen cells, thymus cells, or

PMN, no cytotoxicity was observed.CytotoxicActivities of Other Effector Cells in the Presence

of TAK. The effect of TAK on the cytotoxicitiesof 3 other cellpopulations were examined with MM46 tumor cells as targets.With a casein-induced macrophage fraction (about 80% macrophages)collected in the peritoneal exÃºdate5 days after injectionof casein, cytolysis was maximal at a higher E:T ratio of 50, butthe cytotoxic activity was lower than that of casein-induced PMN(Chart 5). Normal resident peritoneal cells, which consisted of65% macrophages and 35% lymphocytes, and J774 cells (atransformed macrophage-like cell line) did not lyse MM46 tumorcells at any dose of TAK or E:T ratio tested, with TAK at 0.1 to100 f/g/ml and E:T ratios of 0.8 to 200. Resident peritoneal cellsshowed cytotoxic activities in the presence of anti-MM46 antibody and wheat germ agglutinin, and J774 showed cytotoxicityonly with wheat germ agglutinin (Table 3).

Effect of Linear0-1,3-Glucans with VariousChain Lengths.The minimum molecular weight of TAK required for induction ofPMN cytotoxicity against MM46 tumor cells was investigated bytesting the effects of 8 TAK derivatives with different molecularweights (Table 4). Glucans with a DPN of 125 or more inducedPMN cytotoxicity, whereas those with a DPN of 50 or less wereineffective. Four effective glucans showed similar dependencieson the E:T ratio and gave similar dose responses (data notshown) for induction of PMN cytotoxicity. CM-TAK had no effect.None of these TAK derivatives had any direct cytotoxicity onMM46 tumor cells.

DISCUSSION

Here, we have shown that mouse PMN can destroy tumortarget cells in the presence of the antitumor polysaccharideTAK(12, 25). The cytotoxic activity of PMN induced by TAK wasextremely strong, resulting in almost 100% cytolysis of targetcells at an E:T ratio as low as 3 (Chart 1). Similar cytotoxicactivity was obtained when PMN were stimulated with P. acnes,BCG, zymosan A, and Nocardia CWS but not with 23 otherimmunomodulators and agents (Table 1). Thus, the immunomodulators could be divided into those that induced strong PMNcytotoxicity and those that induced none. With all the effectiveimmunomodulators, PMN showed similar cytotoxicity, which wasmaximal at E:T ratios of 3 to 50 (Chart 4).

Antibody, lectin, phorbol ester, etc., have been reported toactivate PMN (6,9,19, 20, 27-29), and PMN stimulated by PMAwere shown to be capable of destroying tumor cells (19,20,28).In our assay system, PMA induced the same cytolytic pattern asdid the effective immunomodulators (data not shown). PMA andTAK are both chemically defined substances and induce thesame potent PMN cytotoxicity resulting in almost 100% cytolysis


1498




Table 1Effects of various immunomodulators and other agents on PMN cytotoxicity against MM46 tumor cells

PMN and 51Cr-labeled MM46 tumor cells were incubated at an E:T ratio of 12:1 in the presence of the indicated compounds (100 jig/ml) at37 Â°Cfor 16 h. Then, the radioactivity of the supernatant was determined.

% ofcytolysisImmunomodulatorTAKLentinanCM-TAKYeast

mannanSchizophyllanTC13BCGP.

acnesZymosanANocardiaCWSE.

coliViableHeat

killedOK432LJpopcriysaccharidePolyinosinic-polycytidylic

acidMuramyl

dipeptideBestatinLevamisole+

PMN96.7Â±2.2s-2.0Â±2.6-1.6

Â±0.4-9.0Â±3.10.9Â±3.03.0+2.397.4

Â±1.9101.6Â±9.099.2

Â±8.695.4Â±2.60.2

Â±1.0-2.3Â±2.222.3Â±3.23.6

Â±1.10.9Â±2.13.9

Â±2.32.0+3.06.9Â±4.9-PMN1.4+1.90.6

Â±1.91.7+0.40.7Â±6.20.2Â±4.0-1.2

Â±1.9-0.4

Â±6.9-1.0Â±0.78.7

Â±3.84.8Â±3.9-5.5

Â±1.10.8Â±4.413.0+2.23.3

Â±1.5-1.5Â±1.34.9+1.2-4.1

Â±0.22.8Â±0.9Agentf-MLP*Carrageenan

Â»CarrageenanXCaseinGlycogenThioglycolateDextranConcanavalin

ACarbonPolystyrene

latex%

ofcytolysis+

PMN-0.5Â±0.8-5.3Â±2.2-6.2

Â±1.87.5

+2.04.5Â±4.7-1.1

Â±4.67.4

Â±1.63.7

Â±4.61.1

Â±4.0-5.5Â±2.9-PMN-1.2

+0.52.2Â±0.62.7Â±3.3-2.3

Â±3.50.4Â±2.21

.3 Â±1.82.6

Â±2.64.9

Â±1.40.9

Â±2.31.0Â±1.5

* Values are means for duplicate determinations.6 f-MLP, W-Formyl-L-methionyl-L-leucyl-L-phenylalanine.

Table 2Targefcell specificity of PMN cytotoxicity induced by TAK

Casein-induced PMN and 51Cr-labe!edtarget cells were incubated at an E:Tratio of 12:1 in the presence of TAK (100 Â»ig/ml)at 37 Â°Cfor 16 h.

TargetMM46

MM48MH134EL-4YAC-1Thymus cellSpleencellPMN+

PMN, +TAK103.2Â±0.2*

80.1 +5.994.2 + 5.097.4 Â±6.091.2 + 10.9-6.4 + 1.1-6.0 Â±5.0

-18.7 Â±11.4%

ofcytolysis+

PMN. -TAK-4.0

+ 2.44.1 Â±2.4

-1 .3 Â±1.0-6.7 Â±5.8

4.9 Â±1.3-5.4 Â±1.8

0.8 Â±2.40.0 Â±1.8-

PMN, +TAK-6.4

Â±6.00.1 Â±3.1

-3.3 Â±1.0-5.7 Â±2.1

0.4 Â±0.4-2.4 Â±2.6

-10.0 Â±1.7

Values are means for duplicate determinations.

of target cells at an E:T ratio as low as 3. PMA can elicit PMNcytotoxicity at a lower concentration (100 ng/ml) than that ofTAK, however, ft is very toxic and is a potent tumor promoter(30), whereas TAK is a nontoxic antitumor polysaccharide. Thesefacts suggest that TAK should be more useful.

PMN cytotoxicity induced by TAK was observed with all thetumor cells tested but not with normal cells (Table 2). Thus, PMNcytotoxicity is selective, and as far as tested PMN can distinguishtumor cells from normal cells. However, the possibility that thisselective cytotoxicity is a function of the susceptibility of thetumor cells can also be taken into account, as reported by Nathanef al. (19). The mechanism of this selective toxicity is unknown,but similar selective toxicity has been observed with activatedmacrophages (13,23) and PMN (2,10, 22).

The observed toxicity was very unlikely to be due to macrophages contaminating the PMN fraction, because this fractionconsisted of about 95% PMN, with only 5% contaminatinglymphocytes and macrophages, and cytolysis was observed atvery low E:T ratios. Moreover, a casein-induced macrophage

fraction showed lower cytotoxicity and required a higher E:T

Effector-to-target ratio

Chart 5. Cytotoxicities of the casein-induced macrophage fraction and residentperitoneal cells in the presence of TAK. 61Cr-labeled MM46 tumor cells wereincubated at 37 Â°Cfor 16 h. Â».with the casein-induced macrophage fraction andTAK (100 //g/mi); O, with the casein-induced macrophage fraction and saline; â€¢with resident peritoneal cells and TAK (100 wg/ml); D. with resident peritoneal cellsand saline. Values are means for duplicate determinations.

ratio than did the PMN fraction (Chart 5). Therefore, we concluded that PMN, not contaminating macrophages, cause lysisof tumor cells in the presence of effective immunomodulators.

Preparations of PMN incubated alone and with TAK in thepresence of target MM46 tumor cells showed the same viabilitymeasured by the trypan blue dye exclusion test (data not shown).This suggests that PMN cytotoxicity is due not to dying PMNbut to some active cytotoxic mechanism. 0-1,3-Glucans are

capable of activating the alternative pathway of complement (11,21), but this PMN cytotoxicity did not require the presence ofserum (data not shown). Therefore, a complement-activating

effect of these immunomodulators cannot explain their induction


1499




TablesCytotoxic activity of effector cells against MM46 tumor cells in thepresence of TAK,antibody, and lectin

Casein-inducedPMN, resident peritonealcells, and J774 cells as effector cells were incubated with 51Cr-labeledMM46target cells in the presence of TAK (100 ng/ml), anti-MM46 antibody (20 jig/ml) and wheat germ agglutinin lectin (25 (/g/ml) at 37 Â°Cfor 16 h.

PMN ResidentPC J774 cells

AdditionTAK

Anti-MM46 antibodyWheat germ agglutininNoneE:T

ratio,1299.7

Â±6.0?1.6 Â±0.6

28.7 Â±1.7-2.9 Â±2.7E:T

ratÂ«,5099.6

Â±6.43.9 Â±5.9

50.8 Â±4.2-8.6 Â±3.3E:T

ratio,124.2

Â±4.025.9 Â±1.947.7 Â±1.6

3.1 Â±9.0E:T

ratio,50-2.6Â±1.7

75.1 Â±3.066.1 Â±3.10.4 Â±0.6E:T

ratio,124.1

Â±5.4-0.9 Â±3.832.8 Â±0.7

2.5 Â±2.0E:T

ratio,50-0.6Â±3.0

-2.0 Â±0.534.4 Â±0.2-2.7 Â±1.0

* Values are means for duplicate determinations.

Table 4Effect of linear ÃŸ-1,3-glucans with various chain lengths on PMN cytotoxicity

against MM46 tumor cellsPMN and 51Cr-labeledMM46 tumor cells were incubated at an E:T ratio of 12:1

in the presence of linear /i-1,3-glucans with various chain lengths (100 /ig/ml) at37Â°Cfor16h.

DPN OfTAK540

(TAK)29912550441484540

(CM-TAK)Saline%

ofcytolysis+

PMN -PMN94.8Â±5.0* 2.6Â±1.594.1

Â±3.7 1.3Â±2.587.6Â±6.8 1.2Â±2.40.3Â±0.4 -0.9 Â±1.40.9Â±0.5 -0.6Â±1.0-4.2Â±0.1 2.1 Â±0.10.9Â±6.1 -1.4Â±3.2-1.0Â±1.3 3.2 +0.1-3.2Â±2.2 -2.2 Â±0.9-5.3Â±1.4 0.0 Â±0.1

* Values are means for duplicate determinations

of PMN cytotoxicity. As an alternative possibility, we examinedthe role of active oxygens and found that the cytotoxic mediatorwas hydrogen peroxide."

The present results show that some antitumor immunomodu-

lators induced potent PMN cytotoxicity in vitro. It seemed important to compare the molecular structures of these immuno-

modulators to understand the induction of this PMN cytotoxicity.Among the 28 immunomodulators and other agents thus fartested, TAK, BCG, P. acnes, zymosan A, and Nocardia CWSwere found to be effective. However, the last 4 immunomodulators are microorganisms themselves or crude cell wall preparation, whereas TAK is a purified and chemically defined neutralpolysaccharide (12). Accordingly, the chemical structures of poly-

saccharides were compared with their effects in inducing PMNcytotoxicity. Glycogen and dextran, which are a-glucans, were

not effective. Of the antitumor polysaccharides tested, only TAKwas effective. Lentinan, yeast mannan, schizophyllan (16), andTC 13 (14), which is /3-1,4-gluban with mannosyl branching, werenot effective. TAK, lentinan, and schizophyllan are all 0-1 ,3-

glucans, but they differ in structure. Lentinan (3) and schizophyllan (16) are 0-1,3-glucans with (1,6)-glucopyranoside branching, whereas TAK is a linear 0-1,3-glucan without branching (12).This suggests that linear 0-1,3-glucans are effective. CM-TAK

did not induce PMN cytotoxicity, although it has the sameskeletal structure as does TAK (24). Its ineffectiveness wasclearly >due to the presence of carboxymethyl groups, whichconvert TAK to a soluble and strongly negatively charged molecule. These results suggest that a neutral linear 0-1,3-glucan is

4K. Morikawa, S. Kamegaya,M. Yamazaki, and D. Mizuno. Hydrogen peroxideas a tumoricidal mediator of polymorphonuclear leukocytes induced by a linear ,<-1,3-o-glucanand some other immunomodulators, submitted for publication.

effective for inducing PMN cytotoxicity.We found that linear 0-1,3-glucans with DPN values of 125 or

more induced strong PMN cytotoxic activity, whereas those withDPN values of 50 or less did not (Table 4). Thus, the minimummolecular size of 0-1,3-glucan required for induction of PMN

cytotoxicity appears to be about DPN 125. Potential contributions of the molecular weight and conformational structure ofpolysaccharides to antitumor activity have been suggested fromresults on lentinan and 0-1,3-glucans (25, 26). The requirement

of a certain molecular structure for the induction of PMN cytotoxicity by glucans suggests that there might be some receptormechanism, possibly on the effector cells, and that the cellsrecognize the chemical structure and conformation of the polysaccharide.

In a previous report (17), we compared the PMN-inducing

activities of various immunomodulators and other agents. Weclassified these compounds quantitatively into 2 types based ontheir effects on PMN high and relatively low inducing immuno-

modulators. We also classified them qualitatively into 2 typesbased on the duration of the PMN induction, declining andpersistent. There is a close correlation between the presentimmunomodulator-induced PMN cytotoxicity and the PMN-in

ducing activity reported in the previous paper. All the immunomodulators shown to be effective in this work are high-PMN-

inducing immunomodulators, and TAK and P. acnes in particularare persistent-type immunomodulators. In general, the PMN

response to conventional foreign agents is of the declining type;TAK and P. acnes were the only 2 persistent-type immunomod

ulators among 14 immunomodulators examined in the previousstudy.

From these results, effective immunomodulators not only induce large numbers of PMN as effector cells in vivo but alsoelicit extremely potent PMN cytotoxic activities in vitro. Since theeffective E:T ratio is very low, the effective immunomodulatorspresumably exert antitumor activity in vivo by modulating theactivity of PMN. Actually, i.p. injection of these immunomodulators into mice significantly reduced the establishment of tumors that developed on i.p. injection of MM46 tumor cells.6

Therefore, these immunomodulators should be useful for immu-

notherapy of ascitic tumors.The experiments reported here indicate that PMN are impor

tant and very powerful effector cells for the destruction of tumorcells in cooperation with some immunomodulators. The inductionof PMN cytotoxicity by immunomodulators appears to representa new effector mechanism, namely, PMN-mediated cytotoxicity,

5K. Morikawa, M. Yamazaki,and D. Mizuno, manuscript in preparation.


1500




with respect to the antitumor activity of these Â¡mmunomodula-

tors.

REFERENCES

1. Baldwin, R. W. Immunotherapy of tumors. In: H. M. Pinedo (ed.). CancerChemotherapy 1981, pp. 178-202. Amsterdam: Excerpta Medica, 1981.

2. Chee, D. D., Townsend, C. M., Gallbraith, M. A., Eiber, F. R., and Morton, D.L. Selectivereduction of human tumor cell populationsby humangranulocytesin vitro. Cancer Res., 38: 4534-4539,1978.

3. Chihara,G. The antitumor polysaccharidelentinan: an overview. In: T. Aoki, I.Urushizaki,and E. Tsubura (eds.), Manipulationof Host DefenceMechanisms,pp. 1-16. Amsterdam: Excerpta Medica, 1981.

4. Chihara,G., Maeda, Y. Y., and Hamuro, J. Current status and perspectivesofimmunomodulatorsof microbial origin. Int. J. Tissue Reac.,4: 207-225,1982.

5. Clark, R. A., and Klebanoff, S. J. Neutrophil-mediatedtumor cell cytotoxicity:role of the peroxidase system. J. Exp. Med., 141: 1442-1447, 1975.

6. Clark, R. A., and Klebanoff, S. J. Role of the myetoperoxidase-HzCyhalidesystem in concanavalin A-induced tumor cell killing by human neutrophils. J.Immunol., Ã•22:2605-2610,1979.

7. Fidter, I. J., Berendt, M., and Oldham, R. K. The rationale for and design of ascreening procedure for the assessment of biological response modifiers forcancer treatment. J. Biol. Resp. Mod., 1:15-26,1982.

8. Fisher, B., and Saffer, E. A. Tumor cell cytotoxicity by granulocytes fromperipheral Wood of tumor-bearing mice. J. Nati. Cancer Inst., 60: 687-691,1978.

9. Gale. R. P., and Zighelboim, J. Polymorphonuclear leukocytes in antibody-dependent cellular cytotoxicity. J. Immunol., 114:1047-1051,1975.

10. Gerrard, T. L, Cohen, D. J., and Kaplan, A. M. Human neutrophil-mediatedcytotoxicity to tumor cells. J. Nati. Cancer Inst., 66: 483-488,1981.

11. Hamuro, M.. Rollinghoff, M., and Wagner, H. Induction of cytotoxic peritonealexÃºdatecells by T-cell immune adjuvants of the -Â¡(1-3)glucan-type lentinanand its analogues. Immunology,39: 551-559,1980.

12. Harada, T., Misaki, A., and Saito, H. Curdlan: a bacterial gel-forming 0-1,3-glucan. Arch. Biochem. Biophys., 724: 292-298,1968.

13. Hibbs, J. B., Jr. Discrimination between neoplasie and nonneoplasticcells invitro by activated macrophages.J. Nat).Cancer Inst., 53: 1487-1492,1974.

14. Inoue, K., Kawamoto, K., and Kadoya, S. Structural studies on an antitumorpolysaccharide from Microellobosporia grÃsea.Carbohydr. Res., 114: 245-256.1983.

15. Inoue, T., and Sendo, F. In vitro induction of cytotoxic polymorphonudearleukocytes by supernatant from a concanavalinA-stimulated spleen cell culture. J. Immunol., 131: 2508-2514,1983.

16. Kikumoto, S., Miyajima, T,, Kimura, K., Okubo, S., and Komatsu, N. Polysaccharide produced by Schizophyllum commune. Part II. Chemical structure ofan extracellular polysaccharide. Nippon Nogeikagaku Kaishi, 45: 162-168,1971.

17. Morikawa, K., Kikuchi,Y., Abe, S., Yamazaki,M., and Mizuno, D. Earlycellularresponses in the peritoneal cavity of mice to antitumor immunomodulatorsGann, 75: 370-378,1984.

18. Nakayama, M., Sendo, F., MiyakÃ©,T.. Fuyama, S.. Arai, S., and Kobayashi,H. Nonspecific inhibition of tumor growth by allo-sensitized lymphocytes. J.Immunol.. 120.:619-623,1978.

19. Nathan,C. F.. Brukner, L. H., Silverstein,S. C.. and Conn, Z. A. Extracellularcytolysis by activated macrophages and granulocytes. I. Pharmacologietriggering of effector cells and the release of hydrogen peroxide. J. Exp. Med.,149: 84-99, 1979.

20. Nathan, C. F.. Silverstein,S. C., Brukner, L. H., and Cohn, Z. A. Extracellularcytolysis by activated macrophagesand granulocytes. II. Hydrogen peroxideas a mediator of cytotoxicity. J. Exp. Med., 749:100-113,1979.

21. Okuda, T., Yoshioka, Y., Ikekawa, T., Chihara,G., and Nishioka, K. Anticom-plementaryactivity of antitumor polysaccharides.Nat. New Biol., 238: 59-60,1972.

22. Pickaver,A. H., Ratdiffe, N. A., Williams,A. E., and Smith, H. Cytotoxic effectsof peritonealneutrophils on a syngeneic rat tumor. Nat. New Biol., 235: 186-187, 1972.

23. Piessens,W. F., Churchill, W. H., and David. J. R. Macrophagesactivated invitro with lymphocytemediatorskiHneoptasticbut not normalcells.J. Immunol.,774:293-299,1975.

24. Sasaki,T., Abiko, N., Nitta, K., Takatsuka, N., andSugiriÃ³,Y. Antitumoractivityof carboxymethylglucansobtained by carboxymethylation of (1-3)-,i-o-glucanfrom Alcaligenesfaecalisvar. myxogenesIFO 13140.Eur. J. Cancer, 75:211-215,1979.

25. Sasaki, T., Abiko, N., Sugino, Y., and Nitta, K. Dependenceon chain length ofantitumor activity of (1 â€”Â»3)-/J-o-glucanfrom Alcaligenes faecalis var. myxogenes. IF013140, and its acid-degradedproducts. Cancer Res. 38:379-383,1978.

26. Sasaki, T., Takasuka, N., Chihara,G., and Maeda, Y. Y. Antitumor activity ofdegraded products of lentinan;its correlationwith molecularweight. Gann,67:191-195,1976.

27. Simchowitz, L., and Schur, P. H. Lectin-dependentneutrophil-mediatedcytotoxicity. I. Characteristics. Immunology,37: 303-311,1976.

28. Tsunawaki, S., Ikenami, M., Mizuno, D., and Yamazaki, M. Mechanisms oflectin and antibody-dependent polymorphonudear leukocyte-mediatedcytolysis. Gann, 74: 265-272,1983.

29. Tsunawaki, S., Oshima, H., Mizuno, D., and Yamazaki, M. Induction of poly-morphonuclear leukocyte-mediated cytolysis by wheat germ agglutinin andantitumor antibody. Gann, 74: 258-264,1983.

30. Weinstein, I. B., and Troll, W. National Cancer Institute workshop on tumorpromotion and cofactors in carcinogenesis. Cancer Res., 37: 3461-3463.1977.

31. Yamazaki, M., Shinoda, H., and Mizuno, D. Co-operation between macrophages and a factor from lymphocytes in tumor lysis in vitro. Gann, 66: 489-497,1975.

32. Yamazaki,M., Shinoda,H., and Mizuno, D. Antibody-dependentmacrophage-mediatedcytolysis in a murine syngeneictumor system. Gann. 67: 651-660,1976.


1501



1985;45:1496-1501. Cancer Res Kaoru Morikawa, Reiko Takeda, Masatoshi Yamazaki, et al. Immunomodulators in Murine Cells

-1,3-d-Glucan and OtherβLeukocytes by a Linear Induction of Tumoricidal Activity of Polymorphonuclear

Updated version

http://cancerres.aacrjournals.org/content/45/4/1496

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/45/4/1496To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



induction of tumoricidal activity of …...rides tested, a linear /3-1,3-glucan structure and a...

Documents