CELLULAR

IMMUNOLOGY

idd,358-366

(1992)

Tumor Cytostasis Mediated by LPS- or PSK-Activated Human Plastic-Adherent Peripheral Blood Mononuclear Ceils YOSHIROKOBAYASHI,' TAKASHI YOSHIKAWA,

AND NAOKO WATANABE

Department of Biomolecular Science, Faculty of Science, Toho University, 2-1, Miyama 2 chome, Funabashi, Chiba 274, Japan Received May 22, 1992; accepted June 29, I992

We investigated the mechanism of cytostasis mediated by activated human plastic-adherent peripheral blood mononuclear cells (PBMC) in two cell lines, L. P3 cells (TNFol sensitive) and A375 cells (TNFol insensitive), using two biological responsemodifiers, lipopolysaccharide (LPS) and a protein-bound polysaccharide extracted from a fungus, PSK. In L. P3/LPS, L. P3/PSK, and A375/LPS cultures, the cytostatic effectswere significantly reversed by anti-TNFa antibody, while in the A375/PSK culture they were not. In concordance with this, LPS was a good inducer of TNFol, but PSK was not. In A375/PSK culture, PSK-activated cells arrested A375 cells at the boundary between Gl and S, presumably through inhibition of polyamine synthesis.This growth inhibition may be mediated by an unknown soluble factor which is different from TNFol, IL- 1, IL-6, and TGF@. 0 1992 Academic press, hc. INTRODUCTION

Activated monocytes/macrophages are known to suppress the proliferation of a variety of tumor cells in a contact-dependent or -independent manner. Several mediators have been reported to be responsible for contact-independent growth inhibition by activated monocytes/macrophages.These include TNFa (1), IL- 1 (2), active oxygen (3), nitric oxide (4), and arginase (5). In determining which mediator(s) is responsible for the growth inhibition, both target cell type and stimulant appear to be crucial. For instance, when TNFa-susceptible cells, e.g., WEHI 164 cells, were used as target cells, TNFa was the mediator found to be mainly responsible for their lysis by activated macrophages (1). With regard to stimulants, nitric oxide production was reported to require both LPS2and interferon-y (IFIVy) (6), while TNFa was produced in response to LPS alone, suggestingthat stimulants play a role in the determination of responsible mediator(s). PSK, a protein-bound polysaccharide extracted from the fungus Corioulous versicolor, has been usedas a biological responsemodifier in the treatment of cancer patients in Japan (7). Although the antitumor mechanism of PSK is not fully understood, a recent study has demonstrated that this agent induced the mRNA expression of a ’ To whom correspondence should be addressed. * Abbreviations used:PBMC, peripheral blood mononuclear cells; LPS, lipopolysaccharide;ODC, omithine decarboxylase; IPN-y, interferon-y; MCAF, monocyte chemotactic and activating factor; FCS, fetal calf serum. 358 00088749/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

TUMOR CYTOSTASIS BY GROWTH INHIBITORY

FACTORS

359

variety of inflammatory cytokines, including IL-l, IL-6, IL-8, TNFa, and monocyte chemotactic and activating factor (MCAF) (8). Some of these cytokines, e.g., IL- 1 and TNFa, have been suggestedto be mediators in contact-independent tumor cell growth inhibition by activated monocytes/macrophages (1, 2). Therefore, it is possible that LPS- and PSK-activated human plastic-adherent PBMC sharethe mechanism of tumor cell growth inhibition. In this study, we have examined the possibility, using two cell lines, L. P3 cells (TNFa sensitive) and A375 cells (TNFa insensitive), as target cells. Here, we demonstrate that LPS-activated human plastic-adherent PBMC inhibited tumor cell growth through TNFa, and that PSK-activated human plastic-adherent PBMC inhibited tumor cell growth through TNFa or unknown factors in a target cell type-dependent manner. MATERIALS AND METHODS Reagents and Cells LPS (Escherichia coli, 055:B5) was purchased from Difco Laboratories (Detroit, MI). PSK was kindly provided by Kureha Chemical Ind. Co. Ltd. (Tokyo, Japan). Rabbit anti-human TNFa! antibody was kindly provided by Dainippon Pharmaceutical Co. Ltd. (Osaka, Japan). This was prepared by using recombinant human TNFa and was shown to have a neutralizing titer of 4864 for TNFa (100 U/ml). Recombinant IL-la was also kindly provided by Dainippon Pharmaceutical Co., Ltd. IL-6 and TGF-P were purchased from Genzyme (Boston, MA) and rabbit anti-human IL- 1(Y antibody and rabbit anti-human IL-l /3antibody were prepared by using recombinant materials, as described previously (9). Rabbit anti-TGF-P antibody was purchased from R & D systems(Minneapolis, MN). Blood samples from normal healthy donors were kindly provided by the Funabashi Red Cross Blood Center. Human PBMC were separated from the huffy coat by a Ficoll-Urografin density gradient method (10). Cells were suspended, at a cell density of 5 X lo6 cells/ml, in RPM1 1640 medium containing 2% fetal calf serum (FCS; GIBCO/BRL, Gaithersburg, MD), following which they were incubated in a flat-bottomed 96-well plastic plate (C. A. Greiner und Sohne, Frickenhausen, Germany), in a total volume of 100 ~1, for 1 hr at 37°C. The plates were then rinsed twice with prewarmed RPM1 1640 medium to remove nonadherent cells, and 100 ~1 of RPM1 1640 medium containing 10% FCS was added. This procedure yielded nearly 50% CD14+ cells (monocytes), determined by cytofluorometric analysis with anti-Leu M3 monoclonal antibody (Becton-Dickinson, San Jose, CA). Mouse fibroblast L - P3 cells and human melanoma A375 cells were maintained in RPM1 1640 medium containing 10% FCS, and these were used as target cells for activated human plastic-adherent PBMC. Assay of Cytostasis by Activated Plastic-Adherent PBMC Plastic-adherent PBMC were cultured with lo4 target cells (L- P3 or A375), in a final volume of 200 ~1,in the presenceof various dosesof LPS or PSK for 24 hr. Cells were pulsed with 0.2 PCi of [3H]TdR (2 Ci/mmol, ICN Biomedicals Inc., Costa Mesa, CA) for the last 4 hr of the culture; they were then frozen, thawed, and harvested on glass fiber filters, following which radioactivity was determined with a scintillation counter. In one particular set of experiments described in the text, plastic-adherent

360

KOBAYASHI,

YOSHIKAWA,

AND

WATANABE

PBMC were separatedfrom target cells with a Millicell-HA culture minicup (0.4 pm; Japan Millipore Ltd.). Assay of TNFa Activity TNFcv activity was measuredwith L - P3 cells as target cells, according to a previously reported method (11) that was slightly modified. Briefly, serially diluted samples and actinomycin D, at a final concentration of 1 pg/ml, were added to the preformed monolayers of L. P3 cells in a 96-well plate. This was followed by 20-hr culture. The culture supernatants were then removed with paper towels, and crystal violet staining was performed. Color eluted with ethanol in phosphate buffer was measured with a plate reader. Cell Cycle Analysis with FACScan Cell cycle progression was analyzed with a FACScan according to the detergenttrypsin method described by L. L. Vindelov et al. (12), using DNA cell-cycle analysis software (Ver C; sum of broadened rectangles model). Assay of ODC Activity ODC activity wasassayed,using [3H]omithine (40 Ci/mmol. American Radiolabeled Chemicals Inc., St. Louis, MO) as the substrate, according to the method described by Y. Endo ( 13). Results were expressedas the radioactivity of [3H]putrescine, since ODC converts ornithine to putrescine. Statistical Analysis The statistical significance of differences was determined by using Student’s t test or Van der Waerden’s test (14). RESULTS Cytostatic Efect of Activated Human Plastic-Adherent PBMC To test the possibility that LPS- and PSK-activated human plastic-adherent PBMC share the mechanism of tumor cytostasis, we used two cell lines, mouse fibroblast L. P3 cells (TNFol sensitive) (11) and human melanoma A375 cells (TNFcx insensitive), as target cells. When target cells were cultured with plastic-adherent PBMC in the presence of various doses of LPS or PSK, their proliferation was inhibited in a dose-dependent manner, as shown in Fig. 1. It should be noted here that [3H]TdR uptake by plasticadherent PBMC was not significant, irrespective of the presence or absence of stimulants, suggesting that [3H]TdR uptake reflected the proliferation of target cells. Further, PSK, at a concentration of 1 mg/ml, reduced the proliferation of target cells to 81 f 8% of the control, suggesting that the activated plastic-adherent PBMC inhibited the proliferation of target cells. When anti-TNFcu antibody was included in the culture, at a final concentration of l/500, which was sufficient to neutralize 10,000 U/ml of TNFa, the inhibition of target cell proliferation was significantly reversed, except for the case of A375/PSK. For example, in the case of L * P3/LPS (0.1 ccgfml), 37 k 6% of the control was restored to 86 +- 1% with the antibody. In

TUMOR CYTOSTASIS BY GROWTH INHIBITORY

361

FACTORS

b

10

100 loo0

0

0.01 01

1

d

0

10

CONCENTRATION

loo

1030

0

0.01 0.1

1

(jglrnl)

FIG. 1. Cytostatic effectsof activated human plastic-adherentPBMC. Plastic-adherentPBMC were cultured with target cells (L . P3 or A375) for 24 hr, in the presenceof various dosesof LPS or PSK. Cytostatic effects were evaluated as described under Materials and Methods. The effect of anti-TNFa antibody was also examined. Results are expressedas percentagesof control, taking the radioactivity of nontreated target cells (control) as 100%.Open circles: without antibody; closed circles: with antibody. (a) L +P3 vs PSK-activated cells; (b) L. P3 vs LPS-activated cells; (c) A375 vs PSK-activated cells; and (d) A375 vs LPS-activated cells.

a parallel set of experiments, TNFL~ activity was determined in cultures of plasticadherent PBMC stimulated with LPS or PSK, at 230, 200, and 170 U/ml for 0.0 1, 0.1, and 1 pg/ml of LPS, respectively, and at < 1, 3.5, and 200 U/ml for 0.01, 0.1, and 1 mg/ml of PSK, respectively. Cell Cycle Analysis of A375 Cells Cocultured with PSK-Activated Plastic-Adherent PBMC Since the proliferation of target cells was assayedby [3H]TdR uptake, there is a possibility that cold thymidine, released from the plastic-adherent PBMC, merely blocked [3H]TdR uptake, particularly in the caseof A375/PSK. We therefore performed cell cycle analysis of A375 cells cocultured with PSK-activated plastic-adherent PBMC in order to examine whether or not the growth of A375 cells was actually inhibited by PSK-activated plastic-adherent PBMC or not. FACScan analysis of the cell cycle progression of A375 cells showed that these cells were arrestedat the boundary between Gl and S by coculture with PSK-activated plastic-adherent PBMC, as shown in Fig. 2. Contact-Dependent vs -Independent Growth Inhibition Activated monocytes/macrophages are known to inhibit cell growth both in a contact-dependent and -independent manner. We therefore used a double chamber culture system, a Millicell-HA minicup, to distinguish between these two mechanisms. When a minicup was placed in each well of a 24-well plate, the target cells in the minicup were separated from the plastic-adherent PBMC by a Millipore filter (0.4 wm). Inhi-

362

KOBAYASHI, YOSHIKAWA, AND WATANABE

Mb

m

HO

80

140 C

u

Gl

s

80

110

GM

lb0

FIG. 2. Cell cycle analysis of A375 cells cocultured with PSK-activated plastic-adherent PBMC. Cell cycle progression was analyzed with a FACScan, according to the detergent-trypsin method (see Materials and Methods).

(a) A375 (b) A375JPBMC (c) A375/PBMC/PSK

GI

S

G2/M

13 k 3.9% 22 + 3.3% 43 f 1.2%

41 + 0.7% 40 f 1.1% 24 _+0.5%

46 + 2.2% 38 t 4.1% 32 t 1.5%

bition of the growth of target cells was not changed by this culture system (as shown by the results in Table I), suggesting that, in all the culture combinations, namely L - P3/LPS, L. P3/PSK, A375 /LPS, and A375/PSK, soluble factors were responsible for growth inhibition. It should be noted here that the double chamber culture system actually enhanced the growth inhibition of target cells by nonactivated plastic-adherent PBMC, as described in the footnotes to Table 1 (see Discussion). We tested to determine whether the well-characterized cytokines, IL-I LU(up to 10 U/ml), IL-6 (up to 100 U/ml), and TGF-/I (up to 5 rig/ml), inhibited the growth of A375 cells after a l-day incubation; however, we found no growth inhibition (data not shown). Further, we tested to determine whether anti-IL-la antibody, anti-IL- lp antibody, a mixture of these two antibodies, or anti-TGF-/3 antibody blocked the inhibition ofgrowth of A375 cells by PSK-activated plastic-adherent PBMC; we found no effects on growth inhibition (data not shown).

TUMOR CYTOSTASIS BY GROWTH INHIBITORY

363

FACTORS

TABLE 1 Inhibition of Cell Growth Using a Double Chamber Culture System Target cells

Stimulant

L*P3

LPS PSK LPS PSK

A375

+ Chamber

- Chamber

100 + 28+ I+ 100 f 412 14+

100 f 45* 33& lOOk 39~ 8+

14” 3 3 5’ 8 0

15b 1 4 4d 6 0

Note. The [3H]TdR uptake of target cells was measured when the cells were cocultured with plasticadherent PBMC in the presenceor absenceof LPS (10 &ml) or PSK (1 mg/ml). Experiments were performed both with and without a double chamber culture system. Results are expressed as percentages of control culture with nonactivated cells. Radioactivity in control cultures was “30,439, b53,718, ‘16,331, and ‘77,518 cpm.

Efect ofArginine or Putrescine on Growth Inhibition by PSK-Activated Plastic-Adherent PBMC When A375 cells and L. P3 cells were cultured in an arginine-depleted medium, only the growth of A375 cells was affected (data not shown). Since increased levels of arginase have been reported to be responsible for growth inhibition by activated macrophages (5) we examined the effect of arginine on the growth inhibition. As shown in Table 2, arginine significantly reversed growth inhibition by both PSK-activated plastic-adherent PBMC and nonactivated plastic-adherent PBMC (P < 0.05 by Van der Waerden’s test). Arginase converts arginine into ornithine, the key precursor for putrescine and polyamine biosynthesis, and thus we also tested the effect of putrescine on growth inhibition. As shown in Table 2, putrescine also significantly reversed growth inhibition by both PSK-activated plastic-adherent PBMC and nonactivated plasticadherent PBMC. BecauseODC is a rate-limiting enzyme for polyamine synthesis and converts ornithine into putrescine, we examined ODC activity in A375 cells to determine whether it was reduced upon treatment with PSK-activated plastic-adherent PBMC or their supernatants. As shown in Table 3, ODC activity in plastic-adherent PBMC, but not in A375 cells, was reduced by PSK. Furthermore, ODC activity in A375 cells and in plastic-adherent PBMC was reduced by A375/PBMC coculture, irrespective of the presence or absenceof PSK; i.e., the effect of PSK was not significant. ODC activity in A375 cells was also reduced by the supernatants from PSK-activated plastic-adherent PBMC, as compared with the control supernatants, as shown in Table 3. DISCUSSION This study demonstrated that both LPS- and PSK-activated human plastic-adherent PBMC inhibited tumor cell growth through soluble factors, namely TNFL~ and/or unknown factors, in a target cell type-dependent manner. L. P3 cells are extremely susceptible to TNFcq and, therefore, it would be expected that, irrespective of stimulants, TNFcx would be mainly responsible for the growth

364

KOBAYASHI, YOSHIKAWA, AND WATANABE TABLE 2

Effectsof Arginine and Putrescine on Growth Inhibition by PSK-Activated Plastic-Adherent PBMC Arginine PSK (mg/ml)

O&4

1nlIV

5mMb

0 1

49* 1’ 18 f 7

50 ic 2 23+ 1

60 t 20 29_+ 3

0 1

31 +o 17+6

36 2 4 25 -t 4

ndd nd

Putrescine PSK (mg/ml)

Old4

0 0.1 0.33 1

41 +o 53 f 2 34 ?I 1 24 3~3

0.4 mA4 502 1* 68 -+ 6** 42 f 2*** 26+ 1

2mM 56-c 72* 49+ 31 f

6** 2* ot 3***

a [3H]TdR uptake in the presence of 1 mM arginine was 95 + 7% of that in A375 cells cultured without adherent cells. b [ ‘H]TdR uptake in the presenceof 5 mM arginine was 113 + 3% of that in A375 cells cultured without adherent cells. ’ Each value is expressed as a percentage of control, taking the radioactivity of nontreated target cells as 100%. d nd, not determined. * P i 0.005 (Student’s t test). ** P < 0.05. *** P