Acidic polysaccharide isolated from Phellinus linteus enhances through

Journal of Ethnopharmacology95(2004)

69–76

Acidic polysaccharide isolated from Phellinus linteus enhances through the up-regulation of nitric oxide and tumor necrosis factor-?from

peritoneal macrophages

Gi-Young Kim a,Gap-Seong Choi a,Sang-Hee Lee b,Yeong-Min Park a,c,?

a Department of Microbiology,Pusan National University College of Medicine,Ami-dong1-10,Pusan602-739,South Korea

b Department of Biological Science,Myongji University,San38-2Namdong,Yongin,Kyunggido,South Korea

c Medical Research Center for Cancer Molecular Therapy College of Medicine,Dong-A University,Pusan,South Korea

Received14July2003;received in revised form13February2004;accepted29June2004

Available online14August2004

Abstract

Medicinal mushrooms are increasingly used to treat a wide variety of disease processes.Aqueous extract from the fruiting body or mycelia of Phellinus linteus has been reported to produce antitumor and immunomodulatory activities in vivo and in vitro.However,the mechanisms underlying its tumoricidal effects are poorly understood.The tumoricidal activity of peritoneal macrophages(PM)cultured with acidic polysaccharide(PL)isolated from Phellinus linteus against B16melanoma cells was enhanced in a dose-dependent manner;growth inhibition increased4-fold with200?g/ml of PL.To further characterize the mechanisms of PL,we investigated the effects of PL on phagocytosis and the release of nitric oxide(NO),tumor necrosis factor-?(TNF-?)and reactive oxygen intermediates(ROI).To investigate the phagocytosis of PM,the uptake of Dextran(Dex)-FITC between PL-untreated and PL-treated PM was compared.We found some augment in phagocytosis of PL-treated PM compared untreated group.PL stimulated a dose-dependent increase in NO and TNF-?,but not in ROI production in PM. We suggested that PL has cytotoxicity against Yac-1cells through the up-regulation of NO and TNF-?production.Also,PL enhanced the expression of costimulatory molecules,CD80and CD86,and major histocompatibility complex(MHC)molecules II in PM.The ability of PL upon the up-regulation of these surface molecules involved in antigen-presenting processes may,by inference,activate T-cell-mediated immunity against malignant cells in vivo.Taken together,these results suggest that PL act as an effective immunomodulator and enhances the anti-tumoral activity of PM.

?2004Elsevier Ireland Ltd.All rights reserved.

Keywords:Phellinus linteus;Polysaccharide;Macrophages;Nitric oxide;Tumor necrosis factor-?;Tumoricidal activity;Phagocytosis

1.Introduction

Macrophages that are ubiquitously distributed in tissues are derived from precursors in the bone marrow via the monocytes of the peripheral blood and constitute the mononuclear phagocyte system essential for the support of homeostasis and host defense against intracellular parasitic bacteria,pathogenic protozoa and fungi as well as against tumors,especially metastasing tumors(Van et al.,1986). Macrophages occupy a unique niche in the immune system,?Corresponding author.Tel.:+82512407557;fax:+82512432259.

E-mail address:immunpym@pusan.ac.kr(Y.-M.Park).in that they not only can initiate innate immune responses but they can also be effector cells that contribute to the resolution of these responses.Activated macrophages are considered to be the pivotal immunocytes of the host defense against tumor growth(Fidler and Kleinerman,1993).The tumoricidal activity of macrophages is performed mainly through NO and other substances that are similar to those in neutrophils with the exception of TNF-?(Flick and Gifford, 1984).Especially,NO is a short-lived radical that if formed by the inducible enzyme NO synthase(iNOS),and NO is considered to be a central molecule in the regulation of the immune response to tumors(Klostergaard,1993;Paulnock, 1992).

0378-8741/$–see front matter?2004Elsevier Ireland Ltd.All rights reserved. doi:10.1016/j.jep.2004.06.024

70G.-Y.Kim et al./Journal of Ethnopharmacology95(2004)69–76

Phellinus linteus has been used as a traditional medicinal mushroom in North-east Asia for the treatment of vari-ous diseases,including gastroenteric disorder,lymphatic disease and various cancers.It was reported previously that polysaccharides(PL)extracted from Phellinus linteus have the effect of stimulating cell-mediated and humoral immunity,and inhibiting tumor growth and metastasis(Han et al.,1999;Kim et al.,1996).It was also reported that PL has preventive mechanisms against the inhibition of gap junctional intercellular communication by hydrogen peroxide(H2O2)(Cho et al.,2002).

In the present study,the tumoricidal effects of PL were evaluated in B16melanoma cells co-cultured with peritoneal macrophages(PM)stimulated with PL.The studying hy-pothesis followed was that PL might activate PM and the PM has cytotoxicity against B16melanoma cells via NO and TNF-?by PL.We also examined the changes of phagocytosis and surface markers inferred to activate adaptive immunity against malignant cells in vivo by PL.

2.Materials and methods

2.1.Mice

C57BL/6mice were obtained from the Korean Institute of Chemistry Technology(Daejeon,Korea)and were kept in our animal facility for at least1week before use.All mice used were females between6and8weeks of ages.

2.2.Preparation and characterization of PL

PL was isolated from the fruiting body of mushroom Phellinus linteus using ethanol precipitation methods fol-lowed by DEAE-cellulose and gel?ltration chromatography (Kim et al.,2003a,b).The pro?les of gel?ltration revealed that the molecular weight of PL is estimated to be approx-imately15kDa.The main components of PL are polysac-charides that consist of mannose,glucose,and galactose. Analyses by gas chromatography showed that PL is free of lipids.LPS contamination was excluded by the experiments that LPS antagonist polymyxin B did not affect the prolifera-tion and expression of surface molecules of B cells by PL un-der the concentrations that resulted in a complete inhibition of LPS induced that(Kim et al.,2003a,b).Also,endotoxin was assayed under endotoxin free experimental conditions using a limulus amebocytes lysate(LAL)pyrogen Kit(Biowhittaker, Walkersville,MD).The experiments were conducted ac-cording to the manufacturer’s protocol:100?l of standards, PL or controls were mixed with100?l of LAL reagent and incubated for1h at37?C.Each tube was then examined for gelation.The quantity of endotoxin in PL was≤0.01ng/mg.

2.3.Isolation of peritoneal macrophages(PM)

The PM was isolated from C57BL/6mice,6-to8-weeks-old,which had been injected intraperitoneally with3ml of thioglycollate for3days prior to peritoneal lavage with10ml of Hank’s balanced salt solution(Sigma,St.Louis,MO). The collected cells were washed twice with RPMI1640 (Sigma,St.Louis,MO)and then cultured in RPMI1640 containing10%heat-inactivated FBS,1mM glutamine, 100of IU/ml penicillin and0.1mg/ml of streptomycin at a density2×106cells/ml.The cells were allowed to adhere for3h to a24-well culture?ask at37?C in a CO2incubator. Then,the cultures were washed twice with RPMI1640to remove nonadherent cells prior to the addition of1ml of fresh RPMI1640containing10%FBS.The viability of PM was assessed by trypan blue exclusion,and the purity was determined by the examination of double positive cells against murine Mac-3(PharMingen,San Diego,CA,USA) using FACSCallibur(Becton Dickinson,San Jose,CA, USA).Cell preparations were>96.0%viable and contained >94.8%PM(data not shown).

2.4.Tumoricidal activity by PM stimulated with PL

Cytotoxicity was determined by measuring the radioactiv-ity incorporated into B16melanoma cells after co-cultivation with PM for24h.The PM(2×105cells/well)was?rst in-cubated in either the medium alone or in a medium supple-mented with various PL(10,50and200?g/ml)for24h in 96-well plates.PM were with PL for4h and co-cultured with B16melanoma cells(2×104cells/well)for an additional 24h at the ratios of5:1,20:1,50:1and100:1(PM to Yac-1) in the presence1?Ci of[3H]-thymidine(TdR).At the end of the incubation,the cells were harvested using an automatic multiwell harvester,and the amount of radioactivity incorpo-rated into the target cells were counted in a liquid scintilla-tion counter.The percent of[3H]-TdR uptake inhibition was calculated by the following formula:%uptake inhibition= 1?

cpm of experimental group

cpm in the presence of untreated control PM

×100. 2.5.Nitric oxide assay

The amount of stable nitrite,the end product of NO gen-eration by the activated macrophages,was determined by a colorimetric assay.Brie?y,50?C of culture supernatants was mixed with an equal volume of Griess reagent(1%sul-fanilamide,0.1%naphthylethylenediamine dihydrochloride, 2.5%H3PO4).This mixture was incubated at room temper-ature for10min.The absorbance at540nm was read on an SLT-spectra reader(SLT Lab Instruments,Austria).The ni-trite concentration was determined by extrapolation from a sodium nitrite standard curve.

2.6.Analysis of TNF-?

PM in the absence or presence of PL was cultured for24h.The cells were?xed and permeated with the Cyto?x/Cytoperm kit(PharMingen,San Diego,CA,USA)

G.-Y.Kim et al./Journal of Ethnopharmacology95(2004)69–7671

according to the manufacturer’s instructions.Intracellular TNF-?was stained with?uorescein R-phycoerythrin(PE)-conjugated antibodies(PharMingen)in a permeation buffer. The cells were analyzed on a FACSCalibur?ow cytometer with the CellQuest program.Furthermore,TNF-?was mea-sured using ELISA kit(Pharmingen)according to the manu-facturer’s instructions.

2.7.ROI assay

The intracellular levels of peroxide(H2O2)and super-oxide anions(O2?)were measured by a?ow cytometric analysis of cells stained respectively with5?M of dichlorodihydro?uorescein diacetate(DCF/DA)(Molecular Probes,OR,USA)for30min at37?C and with10?M of hydroethidine(HE)(Molecular Probes,OR,USA)for15min at37?C.The ROI converted the non?uorescent DCF/DA and HE into their respective?uorescent end-products and the reaction could be monitored by?ow cytometry(Gorman et al.,1997).Dead cells and debris were excluded from the analysis by the electronic gating of the forward and side scatter measurements.

2.8.Dextran(Dex)-FITC internalization

The PM was resuspended in PBS–5%FBS and cultured at37?C for15min.They were then incubated with1mg/ml of Dex-FITC at37?C for1h4?C or37?C.The reaction was stopped with cold PBS containing5%FBS and0.1% sodium azide.The cells were washed three times with cold PBS–FBS–azide,and analyzed on a?ow cytometer.

2.9.Analysis of surface markers

To evaluate the expression of co-stimulators and major his-tocompatibility complex(MHC),the PM was stained with PE-conjugated antibodies against CD80,CD86,and I-A b after treatment in the absence or presence of200?g/ml of PL for24h.All antibodies were acquired from PharMingen (San Diego,CA,USA)and used according to the manufac-turer’s instructions.Brie?y,the PM was initially incubated for30min at4?C with Fc Block(PharMingen)to avoid non-speci?c binding.Then,the cells were incubated with PE-conjugated monoclonal antibodies(1?g/106cells)at4?C for 30min in the dark,then washed twice in phosphate-buffered saline(PBS)that contained2%FBS and0.01%(v/v)NaN3, and?xed in1%(v/v)paraformaldehyde.The cells were an-alyzed on a FACSCalibur?ow cytometer.

2.10.Mixed lymphocyte reaction(MLR)by PM

PM was pretreated with50?g/ml mitomycin and mononuclear lymphocytes from splenocytes were isolated by Ficoll density gradient.H-2d BALB/c responder spleen lymphocytes(1×106cells/well)were cultured with H-2b C57BL/6inducers PM(5×104cells/well).The cells were plated into96-well?at bottom tissue culture pallets(Fal-con)and stimulated with PL at indicated concentrations for 3days.RPMI media were used as control.Cell proliferation was estimated based on the cellular reduction of tetrazolium salt MTT by the mitochondrial dehydrogenase of viable cells into a blue formazan product that can measure spectoropho-tometically.

2.11.Statistical analysis

The results were expressed as the mean±S.D.of the indi-cated number of experiments.The statistical signi?cance was estimated using a Student’s t-test for unpaired observations.

A P-value of<0.05was considered to be signi?cant.

3.Results

3.1.PL upregulates PM-mediated cytotoxicity

In order to investigate the cytotoxic activity of PL-activated PM,PL-untreated or-treated PM was co-cultured with B16melanoma cells.The untreated PM showed only a slight decrease of[3H]-TdR uptake as compared to a group composed of target cells only.The degree of cytotoxicity was also dependent on the number of PM.As shown in Fig.1, the PM stimulated with PL enhanced cytotoxicity in a dose-dependent manner,increasing4fold with200?g/ml of PL as compared to untreated PM.200?g/ml of PL-treated PM showed75,42,34,and10%inhibition at ratios of effector to target cells of100:1,50:1,20:1and5:1,respectively.These effects were identical with500ng/ml LPS.PM cytotoxicity in our experiment belongs to cell-mediated cytotoxicity. Also,the PL did not show any changes of cell viability in the PM or B16melanoma cells up to200?g/ml(data not shown).Thus,PL is no direct cytotoxicity and capable of stimulating

macrophages.

Fig.1.Dose response of%inhibition of[3H]-TdR uptake in B16melanoma cells co-cultured with PM stimulated with the various concentrations of PL (10,50and200?g/ml)for24h.PL-treated PM washed twice by PBS,and B16melanoma cells were added to PM suspensions.Reaction mixtures were incubated for24h and were labeled with1?Ci of3H-TdR for the?nal16h.

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Fig.2.Production of nitrite in PM by stimulation of PL.(A)This panel shows dose-dependent effects of PL on NO production in PM stimulated with various concentrations of PL(10,50or200?g/ml).(B)This panel shows differential effects of polymyxin B(PB)on NO synthesis induced by PL or LPS in PM.PM(1×106cells/ml)was cultured in24-well culture plate with PL(200?g/ml),LPS(500ng/ml)or polymyxin B(PB,5?g/ml). Following48h incubation at37?C,nitrite levels in the culture medium were assayed using Griess reagent and measuring absorbance at550nm.Results were expressed as means±S.D.of three separate experiments.Signi?cantly different(?P<0.05)from medium alone(CTL).

3.2.PL increases NO production in PM

PL induced NO in PM and the production of NO by PL was increased in a dose-dependent manner up to200?g/ml (Fig.2A).The differences in NO production between the PL-or LPS-treated groups and the controls were statisti-cally signi?cant(P<0.05).The amount of NO produced in response to50?g/ml of PL was similar to the amount induced by LPS(200ng/ml).Also,200?g/ml of PL treat-ment in PM strikingly increased NO production compared with the LPS-treated group(P<0.05).To exclude the pos-sible contamination of endotoxin in PL preparation,the inhibitory effect of polymyxin B(PB)in the incubation medium completely prevented increased NO production by LPS.However,the PL was not affected by PB(Fig.2B). These results indicate that the PL increased NO production in the PM,and that the increased NO production by the PL was not due to the contamination of bacterial

endotoxin.Fig.3.Expression of TNF-?in PM.PM in the absence or presence of 200?g/ml of PL was cultured for24h.Cells were?xed and permeated with Cyto?x/Cytoperm kit(PharMingen).(A),Intracellular TNF-?was stained with PE-conjugated antibodies in permeation buffer.Cells were analyzed with?ow cytometry.(B),In the parallel experiments,culture supernatants were collected for detecting the level of TNF-?by ELISA.?P<0.05vs. PL-untreated control.

Therefore,NO is one of the important effector molecules that shows the cytotoxicity of PM against B16melanoma cells.

3.3.PL enhances TNF-?production in PM

The effect of PL on the production of TNF-?was in-vestigated as shown in Fig.3.The PL-treated PM signif-icantly increased the intracellular TNF-?production in a dose-dependent manner(Fig.3A).The increasing effect of PL-treated PM on intracellular TNF-?expression was inde-pendent of LPS contamination because PB was completely blocking TNF-?production in LPS-stimulated PM,but not PL-treated PM(data not shown).Furthermore,analysis of TNF-?production by ELISA showed only low cytokine lev-els(<150pg/ml)when PM were unstimulated.PL-or LPS-stimulated PM secreted higher concentrations of TNF-?than those of control PM(2341±123and2568±89pg/ml,re-spectively;Fig.3B).There were no remarkable differences in

G.-Y.Kim et al./Journal of Ethnopharmacology 95(2004)69–7673

the concentrations of TNF-?secreted by PL-or LPS-treated PM.Expectedly,PB signi?cantly decreased TNF-?produc-tion in LPS-stimulated PM,but not PL (data not shown).Combined with Fig.2,these data may indicate that PL,like LPS,augments NO and TNF-?production of PL through LPS-independent signal pathway.

3.4.PL stimulates phagocytic activity,but not ROI formation of PM

In order to investigate the effects of ROI,the ROI levels that included peroxide and superoxide anions in the cells treated with PL were measured in PM treated with PL or LPS by ?ow cytometry with DCF-DA and HE as shown in Fig.4.However,after 0.5,1or 3h incubation of 200?g/ml of PL,there were no signi?cant changes in the level of ROI for PL in

PM.

Fig.4.Generation of H 2O 2and O 2?in PM treated with PL.PM (1×106cells/ml)was treated with 200?g/ml of PL at 37?C for indicated times.After treatment,cells were incubated respectively with 5?M DCFH-DA and 10?M HE for 15min at 37?C.Cells were analyzed with ?ow cytometer.Shaded area at 0h corresponds to control cells.Dash lines or thin lines at 0.5–3h represent PL-treated cells,

respectively.

Fig.5.Effect of PL treatment on phagocytotic activity with Dex-FITC of PM.PM (1×106cells/ml)treated with various concentrations of PL (10,50and 200?g/ml)in vitro for 24h.After stimulation of PL,1mg/ml of Dex-FITC was treated in PM and cultured at 37?C for 1h.After 1h,cells were washed of two times with PBS containing 2%FBS and 0.01%sodium azide and resuspended in 1ml of 1%paraformaldehyde before FACS anal-ysis.Upper panel shows the percentages of phagocytic cells (A),and lower panel shows mean ?uorescence intensity (MFI)(B),respectively.CTL was stained as untreated control group for 1h at 37?C.Each column and each bar represents the mean ±S.D.from three independent experiments,respec-tively.

To assess this cytotoxicity,as described in materials and methods,the PM was exposed to Dex-FITC ?uorescent beads at 37?C for 1h and then the phagocytotic activity was assayed by a FACS analysis.As shown in Fig.5,the majority of the cells (>75%)of each group phagocytosed within 1h of incu-bation.Although high phagocytic activity,PL treatment had some increase the phagocytic activity of the PM with regard to the dose of PL.The above results show that phagocytosis were one of the mechanisms for cytotoxicity in PM stimu-lated with PL.

3.5.PL enhances the expression of costimulatory molecules and MHC II

In addition to the above results,we found that the expres-sion of the surface molecules was changed in PM stimulated

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Fig.6.Representative FACS histograms of CD80,CD86,and MHC II ex-pression on untreated or PL-treated PM.This shows isotype control(shaded area),untreated(dash line),and PL-treated PM(thin line).After incubation with200?g/ml of PL for24h,PM(1×106cells/ml)was stained with PE anti-mouse CD80,CD86,or MHC II and analyzed by FACS.Results of three independent experiments,each one with pool of PM treated with PL,are ex-pressed as mean of percentage of positive population(control/PL-treated PM).?P<0.05vs.PL-untreated control.

with PL.Representative FACS histograms in Fig.6A show the up-modulation of CD80,CD86,and MHC II in the PM during PL treatment.A signi?cant difference was observed in the percentage of these markers obtained from the200?g/ml of PL treatment.

3.6.PL regulates MLR by PM

The PL strongly increased the activated populations of the PM.To access the increase of surface molecules,we in-vestigated that the effects of PL on MLR by PM were il-lustrated in Fig.7.With their concentrations ranging within 10–200?g/ml,MLR were signi?cantly https://www.360docs.net/doc/715988226.html,pared with RPMI media control,the lymphocytes proliferation was increased5,18,21%,respectively(RPMI media control served as

100%).Fig.7.MLR response induced by PM stimulated with PL.PM was pretreated with50?g/ml mitomycin and mononuclear lymphocytes from splenocytes were isolated by Ficoll density gradient.H-2d BALB/c responder spleen lymphocytes(1×106cells/well)were cultured with H-2b C57BL/6inducer PM(5×104cells/well).The cells were plated into96-well?at bottom tissue culture plates(Falcon)and stimulated with PL at indicated concentrations for 3days.RPMI media were used as control.Cell proliferation was estimated by MTT assay.?P<0.05vs.RPMI media.

4.Discussion

PL is known to have anti-tumoral activities(Han et al., 1999;Kim et al.,1996).To investigate whether PL directly affects tumor cells,we carried out an MTT assay and a cell cycle analysis after PL exposure.The PL-treated B16 melanoma cells did not show any differences compared to PL-untreated cells in respect to cell cycle arrest and viability (data not shown).The above results suggest that the PL did not exert any direct cytotoxic effect on the B16melanoma cells.Therefore we can assume that the PL activates host immunity including innate and adaptive immune systems by releasing mediators with cytotoxic activity.In order to de-termine the cytotoxic activity of PL-activated PM,the PL-untreated or treated PM was co-cultured with B16melanoma cells.As shown in Fig.1,the PM stimulated by PL enhanced cytotoxicity in a dose-dependent manner,increasing4-fold with200?g/ml of PL as compared to the untreated PM.NO and TNF-?were effect molecules for tumoricidal activity by macrophages.

In the last few years,NO has been recognized as an impor-tant messenger in diverse pathophysiological functions,in-cluding neuronal transmission,vascular relaxation,immune modulation,and cytotoxicity against tumor cells(Lowenstein et al.,1994).NO has been identi?ed as the major effec-tor molecule involved in the destruction of tumor cells by activated macrophages(Moncada et al.,1991;Lorsbach et al.,1993;Duerksen-Hughes et al.,1992).The cytotoxic ac-tion of LPS/IFN-?-activated primary mouse macrophages against guinea pig L10hepatoma or mouse L1210lymphoma cell lines was blocked by N G-methyl-L-arginine(NMA),

G.-Y.Kim et al./Journal of Ethnopharmacology95(2004)69–7675

an inhibitor of NO production,mimicked by NO or acidi-?ed NO2?,and absent in macrophages form iNOS?/?mice (Hibbs et al.,1987;Stuehr and Nathan,1989;MacMicking et al.,1995).Administration of NOS inhibitors to mice has promoted growth of several transplantable tumors(Yim et al., 1993;Farias-Eisner et al.,1994),and melanoma cells trans-fected with iNOS cDNA did not proliferate and metastasize well(Xie et al.,1995).

It has also been demonstrated that macrophages stimu-lated by TNF-?produce NO through the expression of the iNOS gene,and it is thought that the reactive nitrogen in-termediates(RNI)so induced play a signi?cant role in tu-moricidal activity(Lorsbach et al.,1993).TNF-?has been recognized as an important host defense cytokine that affects tumor cells.Furthermore,the induction of NO and TNF-?production and gene expression by activated macrophages can lead to cytotoxic effects on malignant cells(Duerksen-Hughes et al.,1992;Stuehr and Nathan,1989).Because of the pivotal role of NO and TNF-?in the anti-microbial and tumoricidal activities of macrophages,signi?cant effort has been focused on developing therapeutic agents that regulate NO and TNF-?production(Poderoso et al.,1999).Thus, the importance of phagocytes in infections,in?ammatory response and homeostasis has been recognized.As regards homeostatic maintenance,it is now accepted that the neu-roendocrine and the immune systems have a bidirectional communication mediated by shared chemicals,messengers and receptors(Besedovsky and Del Rey,1996).In a previ-ous report describing the ability of various polysaccharides to generate H2O2from PM,particulate?-glucans such as yeast glucans were able to stimulate signi?cant H2O2pro-duction,whereas gel-formingβ-glucans including lentinan (from Lentinus edodes),grifolan(from Grifola frondosa), and Sclerotinia sclerotiorum IFO9395glucan had no trig-gering effect on the PM in vitro(Adachi et al.,1993).NO and TNF-?were investigated in this study to con?rm the possibility that PL might be an immunomodulator,and PL was found to elicit NO and TNF-?but not ROI production. In addition,PL did not stimulate phagocytic activity.These results support the possibility that NO and TNF-?induction by PL may contribute in vivo to its immunomodulatory and anti-tumoricidal activities.Biological response modi?ers are now widely used in cancer immunochemotherapy to potenti-ate therapeutic ef?cacy or to alleviate the toxicity of cytotoxic anti-cancer agents.When we consider the above descriptions, it is highly interesting that PL upregulated CD80,CD86,and MHC II expression in PM.It can be speculated that TNF-?induced by treatment of PL has induced PM to express these molecules in itself as an autocrine factor,and that the up-regulation of these molecules may help the cognate in-teractions between antigen-presenting cells and T-cells for anti-tumoral activities in vivo.

Although macrophage activation by PL is quite similar to that by LPS,some differences are also observed between PL and LPS.The pretreatment of PL with PB does not af-fect either nitrite generation or TNF-?production,while it abolished LS stimulation of macrophages.Interesting,PL is assumed to have different membrane receptors for its biolog-ical activities.Even though the membrane receptor of PL is not determined yet,some membrane proteins are assumed to act as a receptor in macrophages.Possible examples are com-plement receptor3(CR3)and Toll-like receptors(TLRs).β-Glucans can be thought to activate macrophages and NK cells via CR3(Ross and V?e tviˇc ka,1993;Renzo et al.,1991).CR3 appears to have broad speci?city in reacting with mannose or N-acetyl-d-glucosamine other thanβ-glucans(Sallusto et al., 1995).TLRs constitute a mammalian transmembrane protein family and play crucial roles in innate immune recognition (Kopp and Medzhitov,1999).Therefore,we have been inves-tigated as CR3and TLRs for detecting receptors of PL(data not shown).

PL enhanced PM-mediated cytotoxicity in a dose-dependent manner.As shown in this study,PL activated PM and modulated interaction between the tumor and the im-mune cells to enhance anti-tumoral activity.In addition,PL enhanced costimulatory molecules and MHC molecules in PM.Based on these results;we propose that PL is a good im-munotherapeutic and immunomodulatory anticancer agent. In order to investigate the overall anti-tumoral effect of PL, a study on the production of immunomodulatory cytokines and molecules mediating antigen presentation is underway in our laboratory.

Acknowledgement

This study was?nancially supported by grant No. R132********-00302002from the Medical Research Cen-ter for Cancer Molecular Therapy of the Korea Science& Engineering Foundation and by Pusan National University in the program,Post-Doc.2004.

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