h”Zw2Dphcl~QCO@“, 23 (1992) 163- 171 Q 1992 Elsevier Science Publishers B.V. All rights reserved. 0162-3109/92p05.00
163
IMPHAR 00584
Inhibitory effect of bestatin lymphocytes Kazuhiko
Ino I,‘, Ken-ichi
Isobe ‘, Setsuko Goto2, Izumi Naka
and Yutaka Tomoda2 ‘Deparlmenl of Immuno!ogv, *Deparhnenl of i%rewks and Gynecology. NQgOyQhiws~
Schl
of Medic&e, NQ~oYQ.Joprrn
(Received 9August 1991; accepted 18 December 1991)
Abstract: Bestatin at mid to high concentration had inhibitory effect to [‘Hlthymidine incorporation ofhumanlymphocytes. It also decreased the PHA-P, Con A and PWM-induced mitogenicity of human lymphocytes. On the contrary. bestatin had growth stimulatory effect to murine lymphocytes, and enhanced the Con A, LPS-induced mitogenicity of murine lymphocytes. These growth inhibitory (human) and stimulatory (mouse) effect of bestatin was found to be independent of ad&rent cells (macrophage and dendritic cell), indicatory directly working to T or I3 lymphocytes. Key words: Bestatin; [SH]l’hymidine incorporation; Immunosuppressor; T cell proliferation
Introduction
Bestatin (INN : ubenimex), [(2&3R)-3-amino2-hydroxy+phenylbutanoyll-(S)-leucine, is a low molecular weight dipeptide isolated from culture filtrate of Streptomyces otivoreticuti by Umezawa et al. (1976). It is a potent inhibitor of microsomal and cytosotic leucine aminopeptidases and aminopeptidaseB (Leyhausen et al., 1983; Rich et al., 1984). As reported in previous papers, bestatin binds to macrophages, T cells and bone marrow progenitor cells through its binding activity to
Correspondence:K. Ino,Department ofObstetricsandGynecology, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan. AbbreviaGons:Con A, concanavalin A; PBS, fetal bovine serum; LPS, lipopolysaceharide; PHA-P. phytohemagghninin P; PWM, pokeweed mitogen.
aminopeptidases (MUller et al., 1982; Leybausen et al., 1983), and demonstrates a modulating activity of cellular immunity, such as enhancing delayed-type hypersensitivity (Umezawa et al., 1976), T-cell mitogenesis (Ishizuka et al., 1980; Saito et al., 1977), release of interleukin-I and interleukin-2 (Shibuya et al., 1987; Noma et al., 1984), enhancement of allogeneic mixed lymphocyte response (Tahnadge et al., 1986), and augmenting macrophage mediated cytotoxicity (Talmadge et al., 1986; Schorlemmer et al., 1983). Bestatin also shows host-mediated antitumor effects in animal (Abe et al., 1984) and human (Ota et al., 1986) systems in vivo as one of the biological response modiiers (BRMs). Recently we have reported that bestatin had direct growth-inhibitory ef&cts against some kinds of human cancer cell Iines in vitro, such as choriocarcinoma and leukemia (Ino et al., 1991). The aim of the present study is the attempt to
164
elucidate whether best&n shows rnitogenic &ect on various h;lman lymphocytes in vitro as well as on mncine lymphocytes as reported previously, or conversely it might show inhibitory effect on human lymphocytes as shown on human leukemic cells.
Materials and Methods Mice
Speci6c pathogen-free female C3H/HeN mice and BALB/c nu/nu athymic mice, 8-12 weeks old, were obtained from Institute for Laboratory Animal Research, Nagoya University School of Medicine, Japan. Agents Bestatin was generously bided by Nippon Kayaku, Co. Ltd. Tokyo, Japan. Concanavalin A (Con A, Sigma Chemical Co., St. Louis, MO) and phytohemagglutinin P (PHA-P, Sigma) were employed as T cell mitogens; lipopolysaccharide (LPS, Sigma) was employed as a B cell mitogen and pokeweed mitogen (PWM, Sigma) as a T and B cell mitogen. All agents were dissolved in distilled water and filtered through a 0.22 pm millipore filter, then diluted to an appropriate concentration with RPMI 1640 medium {Nissui Phar~aceutic~ Co, Ltd,, Tokyo, Japan) before use. Cell lines
HL-60, a human promyelocytic leukemia cell line (Collins et al., 1977), and MOLT-3, a human T lymphoblastic leukemia cell line (Minowada et al., 1972) were kindly provided by Japanese Cancer Research Resources Bank, Tokyo, Japan. These cell lines were maintained in RPM1 1640 medium (Nissui) supplemented with 10% fetal bovine serum (FBS, Cell Cuhure Laboratories, Cleveland, OH).
Spleens and anuses from mice were minced and squeezed through an 80-mesh steel screen into RPM1 1640 medium to obtain a single cell
suspension. The suspensions of murine spleen cells or thymocytes were washed one time, and diluted to the concentration of 1 x 1O’cellsfml with the same medium containing 10% FBS. Human spleen tissues obtained from two male gastric cancer patients who underwent total gastrectomy with splenectomy at the Nagoya University Hospital were used in this study with their consent. It was confirmed that no invasion of cancer cells was found in these spleen tissues. Human thymus tissues, obtained from an 18.week-fetus delivered from a 24-year-old woman who underwent radical hysterectomy with selective abortion for uterine cervical cancer, were used with her and her husband’s consent according to the guidelines by the Japan Society of Obstetrics and Gynecology. These human spleen and thymus tissues were prepared to single cell suspensions to the final concentr~ion of 1 x 10’ cells/ml by the same procedure as in the murine case described above. Erythrocytes present in spleen cells were removed by Ficoll-Paque (Pharmacia LKB Biotechnology Inc., Piscataway, NJ) gradient. Human peripheral blood lymphocytes were separated from heparinized venous blood freshly taken from healthy donors by Ficoll-Paque gradient, then after washing two times they were suspended in RPM1 164Q medium containing 10% FBS to the final concentration of 1 x IO7cells~ml. Cell viability of all prepared lymphocytes in this study was determined by trypan blue dye exclusion, and the viability was confirmed to be more than 957;. Separation of adherent and non-adherent celLr in murine and human lymphocytes
Some of murine spleen cells prepared as de&bed above were divided to the three groups. The tist group consisted of whole spleen cells without ~atment. In the second group, spleen cells were incu~t~ in a 100 x 20 mm culture dish (Falcon 3003, Becton Dickinson Labware, Lincoln Park, NJ) at 37 “C with 5% CO2 in RPM1 1640 medium with 10% FBS for 2 h. Non-adherent
165
cells were collected by flushing the dish. These ceils contained neither macrophages nor dendritic c&s. In the third group, after spleen c&s were incubated similarly to the second group for 2 h, non-adherent cells were removed by pipetting, and adherent cells were further cultured overnight with fresh culture medium (RPM1 1640 with 10% FBS). Deudritlc cells were collected by pipetting the dish vigorously and they were mixed with some of non-adherent spleen cells obtained from the second group. The treated spleen ceils of both the second and the third groups were re-prepared to the concentration of 1 x 1O’cells/ml before use. fn case of human spleen cells and human peripheral blood l~phoc~e~ we did the same procedure as mu&e spfeen cell separation but omitting the third group.
divided into 96-well microplates to the &al concentration of 1 x lo4 cells/well. After incubation at 37 “C for 72 h or 95 h, [ 3H]~ym~e incorporation assay was done by the same procedure as described above. All experiments were performed in quadruplicate and repeated three times. Relative [ ‘HIthymidine incorporation was calculated by this formula: (cpm in tested w~)~(~prn in control well) x 100 (ye). Backwood ra~oacti~ty was less than SW)cpm in all experiments. Statistical ana&&
data were reported as mean f SD. The significance of differences from the appropriate controls were determined by using Student’s t-test.
All
Results Incorporation of [3H]thymidine Lymphocyte suspensions (1 x lo7 cells/ml) were divided into 96-well flat-bottomed microplates (Falcon 3072, Be&on Dic~mson Labw~e) to the final concentration of 5 x lo5 cells/well. Then various concentrations of bestatin in the presence or absence of mitogens were added into each well to a total volume of 100 ~1. Final concentrations of best&n were 0 (control), 1000,100,14 1.0 and 0.1 ggjml. ~itogens were added at the constant concentration. [The inflations of Con A, PHA-P, LPS, PWM were 5 #jml, 1 pg/ml, 5 pg/ml, 5 #g/ml respectively). Next, plates were incubated at 37 “C for 48 or 72 h in a humidilied atmosphere of 5% CO, and air. The cells were putsed with 0.5 @&‘weU of [~H]~~i~e (~ersh~ Japan, Bun~~ku Tokyo, Japan) during the last 16 h ofculture, then were harvested with a multiple cell harvester (LAB0 MASH, Labo Science Co. Ltd., Tokyo, Japan). The amount of [3H]thymidine into cells was determined by a liquid-scintillation counter (BECKMAN LS7500, Eieckman ~nst~ments, Inc. Fullerton, CA}. Leukemic cells, Ht60 and MOLT-3, suspended in RPM1 1640 medium with 10% FBS at a concentration of 2 x iOs cells/ml, were also
E&ct of bestatin ORDNA sy&w3
of va&us l&mph-
#c9& in visro First we ex~n~ the effects of bestatin on DNA synthesis in murine and human spleen cells and thymocytes in vitro by t3H]thymidine incorporation assay. After treatment of bestatin for 72 h in the absence of mitogens, bestatin significantly enhanced incorporation of [3H]thymi&me into mu&e spleen cells at any ~n~ntrations except loo0 pg/ml. On the other hand, in mmine tbymocytes, bestatin alone did not show any effect on DNA synthesis (Table I). In contrast, bestatin showed growth inhibitory effects on the DNA synthesis of both human spleen cells and thymecytes (Table I). This growth inhibitu~ effect on human l~p~~~ was do~~~d~t. Next, we examined the effects of bestatin on DNA synthesis of the same cells in the presence of mitogens by [3H]thymidine incorporation assay. In this assay, Con A was added at a concentration of 5 ,ug@l when murine cells were cultured, and PHA-P was added at a ~ncen~ation of 1 pg/ml when human cells were cultured. As shown in Table II, the incorporation of [ 3H]thymidine into murine spleen ceils was signXcantly increased by 17% to 150% compared with the
TABLE I Effect of bestatin in the absence of mitogens on murine and human spleen cells and thymocytes
Bestatin concentration
Murine cells WC
Control 0 (~~ml)
1000 100 10 1 0.1
8425 f 6731 * 18?2&t 13379f 11399f 12376f
Human cells
-I 545 (100)” 969E ( 79.9) 595Sb (222.9) 2462b (158.8) 1904u(1353) 2178’ (146.9)
%Y
SPC
6837 & 604 (108) 6215 f 640 ( 90.9) 6816 it 1077 ( 99.7) 662.5t 682 ( 96.9) 7145 f 1179 (104.5) 6741 ?: 1199 ( 98.6)
126362 1857 + 55722 9932 + 11195f 12914 f
Thy 992 (100) 20$ ( 14.7) 117c ( 44.1) 596” ( 78.6) 291” ( 88.6) 852 (102.2)
9263 f 1954i 4456p 6336 f 7355 * 8485 k
996 (100) 27’ ( 21.1) 120’ ( 48.1) 380’ ( 68.4) 647’ ( 79.4) 1086 ( 91.6)
Cells were treated with various concentrations of bestatin for 72 h, then the [3H]thymidine incorporation assay was done. The values represent the mean f SD of three experiments carried out in quadruplicate; values in parentheses &e relative incorporation (%). s Calculated as (cpm in tested welt)/(cpm in control well) x 100 (X) b Si8nilkant increase compared to control (0 pg/ml of bestatin) by Stndent’s z-test (p < 0.05). E Significant decrease compared to control ( p < 0.05).
TABLE II Bffect of bestatin in the presence of mitogens on mu&e and human spleen cells and thymocytes Bestatin concentration
Control 0 (F&ml) 1000 too 10
1 0.1
Murine cells (Con A addedb)
Humancells (PITA-P added=)
SPC
Thy
SPC
Thy
30610 2 4662 (100)” 3918 & 129’ ( 12.8) 76647 f 16096d (250.4) 43282 + 3203d (141.4) 35844 f 4086“ (117.1) 39946 + 399Sd (138.5)
23052 f 1208 (100) 7169 f 222’ ( 31.1) 46081 it 7926d (199.9) 30936 f 43934 (134.2) 22061 rs 2008 f 95.7) 22752 i 1820 ( 98.7)
45420 f 1791 (loo) 7494 + 135” ( 16.5) 22574 f 406= ( 49.7) 41196 ?: 1565“ ( 90.7) 44739 f 761 ( 98.s) 45920 f 1561 (iOl.1)
34587 k 1607 (loo) 31405 + 1916’ ( 99.8) 31025 it. 127T ( 89.7) 30882 f 2653” ( 89.2) 34864 f 6206 (l~.S) 35555 f 4124 (1~2.8~
Cklls were treated with various eoncentrat~ns of bestatin with mitogens for 72 h, then the [sH~th~i~ne ~co~~tion assay was done. The values represent the mean + SD of three experiments carried out in qmtdruplicatc; values in parentheses pive relative incorporation (%). * Calcuiated as (cpm in tested well)/(cpm in control well) x 100 (%) b 5 flg/rnl of Con A was ad&d into each well. c 1 pg/ml of PHA-P was added into each well. * Significant increase compared to control (mitogen alone) by Student’s t-test (p c 0.05). o Significant decrease compared to control (p < 0.05).
control, Con A alone. Namely, be&&in stimulated mitogenicity of Con A on murine spleen cells. 1000 pg/ml of bestatin, a very high dose, may probably exert a ~~oto~c effect on spleen cells. Bestatin also signifkantly stimulated mitogenicity of Con A on murine thymocytes at 100 to
10 pgfml. In addition, by using 1 or 10 pg/ml of Con A, similar stimulator effects of bestatin on mutine cells were observed (data not shown in this paper). On the contr~y, mitogeni~ity of PI-IA-P on human spleen cells and thymocytes was inhibited by more than 10 pg/ml of bestatin
after 72 h treatment,
but bestatin less than 1 pg/ml, clinically used concentrations, showed no effect on mitogeniciry of PHA-P (Table II). Similar results were obtained in the case of 48 h treatment of bestatin in the presence of PHA-P, PWM or Con A (Fig. 1). In order to determine whether bestatin exerts its effect on only T lymphocytes or not, spleen cells from athymic nude mice were cultured with bestatin for 48 h or 72 h in the absence or presence of 5 p&/ml of LPS as a I? cell mitogen. As shown in Pig. 2, the incorporation of [ 3H]thymidine into athymic mouse spleen cells was significantly increased by 72 h treatment of 100 to 1000 pgjmi ofbestatin in the absence of LPS, and mitogenicity rf LPS was also enhanced by the combination treatment of 100 to lOOO&ml of bestatin compared with the LPS alone. Almost similar results were found after 48 h bestatin treatment (data not shown). These resuit may
0
loo00
I’H
zoo00
sooon
&mo
0
Fig. 2. Etkt of bcstatin in the absence or presence of LPS on athymic mouse spleen cells. Cells were treated for 72 h, then the [‘H]thymidine incorporation assay was done. L’ach column and bar represent the mean and SD of three exp& ments. ’ Significant increase compared to control (p < 0.05). b Significant increase compared to LPS alone (p c 0.05).
suggest that bestatin activated the DNA synthesis of murine B cells at a very high concentration, more than 100 pg/ml. Additional experiments by the same assay system were performed with human peripheral blood lymphocytes (PBL). As shown in Fig. 3, mitogenicity of PHA-P on human PBL was inhibited dose-dependently as in the case of human spleen cells, although no significant effect was observed by the treatment of bestatin alone. Eflecctof bestatin on non-aa!herent lymphocytes
Fig. I. Effect of bestath combined with PHA-P, PWM. Con A on human spleen cells. Cells were treated for 48 h, then the [‘Hlthymidine incorporation assay was done. Each column and bar represent the mean and SD of three expcriments. a Significant decrease compared to PHA-P alone by Student’s r-test (p < 0.05). ’ Significant decrease compared to PWM alone (p c 0.05). F Significant decrease compared to Can A alone (p c 0.05).
We examined the etkct of bestatin on three groups of murine spleen cells by the [ 3H]thymidine incorporation assay in order to elucidate whether the mechanism of its effect is related to the existence of adherent cells (macrophages and dendritic ceils). The cell population of each group was described in Materials and Methods. In this assay, bestatin was added at concentrations of 10, 100, 1000 pglml, and cells were treated for 48 h. As shown in Fig. 4, bestatin increased incorporation of [ 3H]thymidine at 10 to 1000 pg/ml in the absence of Con A in a!1 three groups. In the presence of 5 pgjml of Con A, bestatin also stimulated
168 mitogenicity of Con A in all groups at 100 to 1000 pgj’ml. These results suggested that bestatin
PtlAt3sl0 Pth3s1.0
Fig.3. Effect of bestatin in the absence or Presence of PHA-P on human peripheral blood lymphocytes. Cells were treated for 48 h, then the [‘H]thymidine incorporation assay was done. Each Golumnand bar represent the mean and SD of three ex~riments. a S~~j~t decrease compared to PHA-P alone by Student’s t-test ( p e 0.005).
Fig. 4. meet of bestatin on three groups of murine spieen cells. AUgroups were treated with various concentrations of bestatin in the absence or presence of Con A for 48 h, then the [“W]thymidineincorpordion assay was done. Each column and bar represent the mean and SD of three experiments.
showed the enhancing e%ct on the DNA synthesis of murine spleen cells by acting directly on T cells. Next, we examined the effect of bestatin on human non-adherent spleen celis and peripheral blood lymphocytes (PBL) by the same procedure as murine cases. In this assay, bestatin was added at concentrations of ~.l-lOOO~g~m1 in the absence or presence of PHA-P, Bestatin inhibited the DNA synthesis of human non-adherent spleen cells and also inhibited the PHA-induced mitogenicity of them (Fig. 5). Similarly, it inhibited the PHA-induced mitogenicity of human nonadherent PBLs (data not shown). E&et of best&in on DNA synthesis of human leukemic cells in vitro
The same assay was performed with HL-60, a human p~myeloc~ic leukemia cell line, and MOLT-3, a human T lymphoblastic leukemia cell
Fig. 5. Effect of bestatin in the absence or presence of PHA-P on human non-adherent spleen cells. Cekiswere treated for 48 h, then the [‘Hlthymidine incorporation assay was done. Each column and bar represent the mean and SD of three experiments. ” Significant decrease comp-ed to control (P-C 0.05). b Signihcanl decrease compared 10 PHA-P alone (p 4 OM).
I69 TABLE III Effect of bestatin on leukemic cells, HL-60 and MOLT-3 Bestatin concentration
Control 0 (&nl) loo0 100 10 I 0.1
HL-60
MOLT-3
72 h
96 h
loo;’ 0.8 f O.lb 14.7 f 4.9b 48.8 f 14.1’ 74.5 f 10.6’ 93.7 f 7.3’
IO0 1.5 f 10.8 f 29.7 f 67.2 + 91.1 +
0.4b 1.3b 2.4b 2.P 3.T
72 h
96 h
100 42.9 +_1.P 37.1 * 2.P 65.3 f 2.Sb 76.6 + 3.5’ 99.1 f 6.3
IO0 56.6 f 4.4b 28.3 + 2.3b 45.9 + 2.7b 83.4 +-4.6b 104.6 + 7.2
Cells were treated with various concentrations ofbestatin for 72 h or 96 h.then the [‘Hlthymidine incorporation assay was done. The values represent the mean k SD (relative %) for quadrupticares from one of three similar experiments. il Calculated as [cpm in tested well)/(cpm in control well) x 100 (%) ’ Significant decrease compared to control (0 &ml of best&n) by Student’s r-test (p c 0.005). c Significant decrease compared to control (p < 0.05).
line in order to compare with the effects of bestatin on lymphocytes as described above. As shown in Table III, bestatin showed inhibitory elects against the DNA synthesis of both HL-60 and MOLT-3 cells after 72 h or 96 h treatment. The growth inhibition of HL-60 cells by bestatin was observed within the wide range of concentrations of 0.1-1000 &ml in a dose dependent manner, while in MOLT-3, the inhibition was observed at the concentration of more than l.Opg/ml of bestatin. Discussion
We show in this paper that bestatin has a growth inhibitory effect on human lymphocytes contrary to growth stimulation on murine lymphocytes. There have been many reports about the immunoenhancing e&t of bestatin, especially that it has been shown to augment T cell mitogenesis. Saito et al. (1977, 1978) showed the stimulatory effect of best&n on the blastogenesis of guinea pig lymphocytes. Ishizuka et al. (1980) showed that bestatin increased [ 3H]thymidine incorporation into murine lymphocytes, and augmented the mitogenicity of Con A or LPS. These results were
consistent with our results in murine spleen cells. However, the effect of bestatin on human lymphocyte proliferation has not been well studied. Morikawa et al. (1989) reported the suppressive e&t of bestatin on the proliferation and differentiation of human B cells in vitro dose-dependently. Their results correlated to our results, but we show that bestatin inhibited the DNA synthesis of both human T and 3 cells. How does bestatin work as an inhibitor to human T cell proliferation and as a stimulator to murine T cell proliferation? Ishizuka et al. (1980) and Weiljmann et al. (1985) showed that bestatin worked to murine lymphocytes via macrophages. However, our results indicate that bcstatin worked directly to murine lymphocytes to enhance ceU growth and Con A reactivity. We also found that bestatin worked directly to human lymphocytes to inhibit cell growth and PHA-induced mitogenicity. Recently, several studies have reported on the cell surface aminopeptidase activities of both human and murine cells. Amoscato et al. (1989) demonstrated that leucine aminopeptidase-like activity existed on the membrane of human lymphocytes and was inhibited by bestatin. Look et al. (1989) and Ashmun et al. (1990) found that cell surface glycoprotein CDI3, which
was exxpressedan human mon~~es, granuloq&es and myeioid hmkemia ceh lines, was identical to ~o~~t~dase N, and that this enzyme activity was inhibited by bestatin. On the other hand Gorvel et al. ( 1990) and Vivier et al. ( 1941) demonstrated that mouse thymocyte-activating molecule (THAM) was associated with an aminopeptidase N activity and was the counterpart of the hnman CD26 (di~~t~dyi peptidase XV).From these recent report% it is suggested that surface ~~ope~~dase c&d play an ~rn~~~t role in cdt activation, but the relation between best&n’s ef&ct and ~~b~ti~n of ~ino~Fti~e is now m&r ~v~~~tion. We recaE the fact that bestatin had a direct growth ~n~b~to~ elect aa;ainst HL-69, MOLT-3 (in this paper and Sakurads et al., 1990) and choriocarcinoma cells (Ino et al,, 1991). It also works as an inhibitor oa epidermal growth factorinduced DNA synthesis in primary cultured rat hiatuses (Takahashi etal., 19g9). These results indicated that best&in had direct grow& inhibitory effect to some kinds of human cancer cdf lines and normal cells. In this paper; we show that best&n works as an ~~os~u~~or to mtine lyrn~h~~es, but not human ~~rnphocy~~ at higher dose than cl&ical use. We and others showed that best&n had growth inhiitory effects to human cancer cell lines. These results encourage us to use bestatin for cancer therapy. However, we have to check the immunosuppressive effect of this drug at high dose.
AA, Atexmder JW, Babeoek GF. Surface an-&mpeptidase activity of human ~~p~c~es. f lmmtmoi 1989, 142: 1245. Asbmun RA, Look AT, Metalloprotease activity of CD13/aminopeptidase N on the surface of human myeloid cells. Blood 1990: 75: 462. Collins SJ. Gal10RC,Gallaghor RR. Continuous growth and
~j~erenti~tion of ruin myebid feukaemic ccfls in suspension etdtlJm Natum 1977;27a: 347. Gorvei JP, Vivier I, Naquet F, ~r~e~~s P, Rigat A, Pierms M. &haracteri~~n ofthe neutrai arn~~~~t~d~s~ activity associated to the mouse thymocytr?~a&ivating motecutc. J Immunol 1990; 144: 2899. XnoK, Gato S, Kosaki A, Nomura S, Asada E, Misawa T, Furtrhashi Y, Mizutani S, Tomoda Y. Growth inhibitory affect of bcstatin on choriocarcinoma ceil lines in vitro. Biuth~rapy 1991; 3: 351. Ishizuka M, Sate J, Su~yama Y. Takeuehi T, Umezawa H. Mitugenie effect of bestatin on lympkocytes, J Anti&t 1980; 33: 653.
~yba~~~~ 6, Schuster DK, Vaith P, Z&n RK, Umsaawa H, PaIke D, Muher WEG. Identification and properties afthe celi membrane bound ieucina aminopeptidase interacting with the potential immunastimulant and chemmotbsrapeuticagent bestatin. Biochem Pharmacol 1983; 32: 10$1. ~aok AT, Ashmun RA, Shapiro LH, Peiper SC. Human my&id plasma ~m~rane glycoprotein CD13 @p150)is identioa:alto ~ino~tid~aN_ J 4% Invest 1989; 83: 1299.
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163
IMPHAR 00584
Inhibitory effect of bestatin lymphocytes Kazuhiko
Ino I,‘, Ken-ichi
Isobe ‘, Setsuko Goto2, Izumi Naka
and Yutaka Tomoda2 ‘Deparlmenl of Immuno!ogv, *Deparhnenl of i%rewks and Gynecology. NQgOyQhiws~
Schl
of Medic&e, NQ~oYQ.Joprrn
(Received 9August 1991; accepted 18 December 1991)
Abstract: Bestatin at mid to high concentration had inhibitory effect to [‘Hlthymidine incorporation ofhumanlymphocytes. It also decreased the PHA-P, Con A and PWM-induced mitogenicity of human lymphocytes. On the contrary. bestatin had growth stimulatory effect to murine lymphocytes, and enhanced the Con A, LPS-induced mitogenicity of murine lymphocytes. These growth inhibitory (human) and stimulatory (mouse) effect of bestatin was found to be independent of ad&rent cells (macrophage and dendritic cell), indicatory directly working to T or I3 lymphocytes. Key words: Bestatin; [SH]l’hymidine incorporation; Immunosuppressor; T cell proliferation
Introduction
Bestatin (INN : ubenimex), [(2&3R)-3-amino2-hydroxy+phenylbutanoyll-(S)-leucine, is a low molecular weight dipeptide isolated from culture filtrate of Streptomyces otivoreticuti by Umezawa et al. (1976). It is a potent inhibitor of microsomal and cytosotic leucine aminopeptidases and aminopeptidaseB (Leyhausen et al., 1983; Rich et al., 1984). As reported in previous papers, bestatin binds to macrophages, T cells and bone marrow progenitor cells through its binding activity to
Correspondence:K. Ino,Department ofObstetricsandGynecology, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan. AbbreviaGons:Con A, concanavalin A; PBS, fetal bovine serum; LPS, lipopolysaceharide; PHA-P. phytohemagghninin P; PWM, pokeweed mitogen.
aminopeptidases (MUller et al., 1982; Leybausen et al., 1983), and demonstrates a modulating activity of cellular immunity, such as enhancing delayed-type hypersensitivity (Umezawa et al., 1976), T-cell mitogenesis (Ishizuka et al., 1980; Saito et al., 1977), release of interleukin-I and interleukin-2 (Shibuya et al., 1987; Noma et al., 1984), enhancement of allogeneic mixed lymphocyte response (Tahnadge et al., 1986), and augmenting macrophage mediated cytotoxicity (Talmadge et al., 1986; Schorlemmer et al., 1983). Bestatin also shows host-mediated antitumor effects in animal (Abe et al., 1984) and human (Ota et al., 1986) systems in vivo as one of the biological response modiiers (BRMs). Recently we have reported that bestatin had direct growth-inhibitory ef&cts against some kinds of human cancer cell Iines in vitro, such as choriocarcinoma and leukemia (Ino et al., 1991). The aim of the present study is the attempt to
164
elucidate whether best&n shows rnitogenic &ect on various h;lman lymphocytes in vitro as well as on mncine lymphocytes as reported previously, or conversely it might show inhibitory effect on human lymphocytes as shown on human leukemic cells.
Materials and Methods Mice
Speci6c pathogen-free female C3H/HeN mice and BALB/c nu/nu athymic mice, 8-12 weeks old, were obtained from Institute for Laboratory Animal Research, Nagoya University School of Medicine, Japan. Agents Bestatin was generously bided by Nippon Kayaku, Co. Ltd. Tokyo, Japan. Concanavalin A (Con A, Sigma Chemical Co., St. Louis, MO) and phytohemagglutinin P (PHA-P, Sigma) were employed as T cell mitogens; lipopolysaccharide (LPS, Sigma) was employed as a B cell mitogen and pokeweed mitogen (PWM, Sigma) as a T and B cell mitogen. All agents were dissolved in distilled water and filtered through a 0.22 pm millipore filter, then diluted to an appropriate concentration with RPMI 1640 medium {Nissui Phar~aceutic~ Co, Ltd,, Tokyo, Japan) before use. Cell lines
HL-60, a human promyelocytic leukemia cell line (Collins et al., 1977), and MOLT-3, a human T lymphoblastic leukemia cell line (Minowada et al., 1972) were kindly provided by Japanese Cancer Research Resources Bank, Tokyo, Japan. These cell lines were maintained in RPM1 1640 medium (Nissui) supplemented with 10% fetal bovine serum (FBS, Cell Cuhure Laboratories, Cleveland, OH).
Spleens and anuses from mice were minced and squeezed through an 80-mesh steel screen into RPM1 1640 medium to obtain a single cell
suspension. The suspensions of murine spleen cells or thymocytes were washed one time, and diluted to the concentration of 1 x 1O’cellsfml with the same medium containing 10% FBS. Human spleen tissues obtained from two male gastric cancer patients who underwent total gastrectomy with splenectomy at the Nagoya University Hospital were used in this study with their consent. It was confirmed that no invasion of cancer cells was found in these spleen tissues. Human thymus tissues, obtained from an 18.week-fetus delivered from a 24-year-old woman who underwent radical hysterectomy with selective abortion for uterine cervical cancer, were used with her and her husband’s consent according to the guidelines by the Japan Society of Obstetrics and Gynecology. These human spleen and thymus tissues were prepared to single cell suspensions to the final concentr~ion of 1 x 10’ cells/ml by the same procedure as in the murine case described above. Erythrocytes present in spleen cells were removed by Ficoll-Paque (Pharmacia LKB Biotechnology Inc., Piscataway, NJ) gradient. Human peripheral blood lymphocytes were separated from heparinized venous blood freshly taken from healthy donors by Ficoll-Paque gradient, then after washing two times they were suspended in RPM1 164Q medium containing 10% FBS to the final concentration of 1 x IO7cells~ml. Cell viability of all prepared lymphocytes in this study was determined by trypan blue dye exclusion, and the viability was confirmed to be more than 957;. Separation of adherent and non-adherent celLr in murine and human lymphocytes
Some of murine spleen cells prepared as de&bed above were divided to the three groups. The tist group consisted of whole spleen cells without ~atment. In the second group, spleen cells were incu~t~ in a 100 x 20 mm culture dish (Falcon 3003, Becton Dickinson Labware, Lincoln Park, NJ) at 37 “C with 5% CO2 in RPM1 1640 medium with 10% FBS for 2 h. Non-adherent
165
cells were collected by flushing the dish. These ceils contained neither macrophages nor dendritic c&s. In the third group, after spleen c&s were incubated similarly to the second group for 2 h, non-adherent cells were removed by pipetting, and adherent cells were further cultured overnight with fresh culture medium (RPM1 1640 with 10% FBS). Deudritlc cells were collected by pipetting the dish vigorously and they were mixed with some of non-adherent spleen cells obtained from the second group. The treated spleen ceils of both the second and the third groups were re-prepared to the concentration of 1 x 1O’cells/ml before use. fn case of human spleen cells and human peripheral blood l~phoc~e~ we did the same procedure as mu&e spfeen cell separation but omitting the third group.
divided into 96-well microplates to the &al concentration of 1 x lo4 cells/well. After incubation at 37 “C for 72 h or 95 h, [ 3H]~ym~e incorporation assay was done by the same procedure as described above. All experiments were performed in quadruplicate and repeated three times. Relative [ ‘HIthymidine incorporation was calculated by this formula: (cpm in tested w~)~(~prn in control well) x 100 (ye). Backwood ra~oacti~ty was less than SW)cpm in all experiments. Statistical ana&&
data were reported as mean f SD. The significance of differences from the appropriate controls were determined by using Student’s t-test.
All
Results Incorporation of [3H]thymidine Lymphocyte suspensions (1 x lo7 cells/ml) were divided into 96-well flat-bottomed microplates (Falcon 3072, Be&on Dic~mson Labw~e) to the final concentration of 5 x lo5 cells/well. Then various concentrations of bestatin in the presence or absence of mitogens were added into each well to a total volume of 100 ~1. Final concentrations of best&n were 0 (control), 1000,100,14 1.0 and 0.1 ggjml. ~itogens were added at the constant concentration. [The inflations of Con A, PHA-P, LPS, PWM were 5 #jml, 1 pg/ml, 5 pg/ml, 5 #g/ml respectively). Next, plates were incubated at 37 “C for 48 or 72 h in a humidilied atmosphere of 5% CO, and air. The cells were putsed with 0.5 @&‘weU of [~H]~~i~e (~ersh~ Japan, Bun~~ku Tokyo, Japan) during the last 16 h ofculture, then were harvested with a multiple cell harvester (LAB0 MASH, Labo Science Co. Ltd., Tokyo, Japan). The amount of [3H]thymidine into cells was determined by a liquid-scintillation counter (BECKMAN LS7500, Eieckman ~nst~ments, Inc. Fullerton, CA}. Leukemic cells, Ht60 and MOLT-3, suspended in RPM1 1640 medium with 10% FBS at a concentration of 2 x iOs cells/ml, were also
E&ct of bestatin ORDNA sy&w3
of va&us l&mph-
#c9& in visro First we ex~n~ the effects of bestatin on DNA synthesis in murine and human spleen cells and thymocytes in vitro by t3H]thymidine incorporation assay. After treatment of bestatin for 72 h in the absence of mitogens, bestatin significantly enhanced incorporation of [3H]thymi&me into mu&e spleen cells at any ~n~ntrations except loo0 pg/ml. On the other hand, in mmine tbymocytes, bestatin alone did not show any effect on DNA synthesis (Table I). In contrast, bestatin showed growth inhibitory effects on the DNA synthesis of both human spleen cells and thymecytes (Table I). This growth inhibitu~ effect on human l~p~~~ was do~~~d~t. Next, we examined the effects of bestatin on DNA synthesis of the same cells in the presence of mitogens by [3H]thymidine incorporation assay. In this assay, Con A was added at a concentration of 5 ,ug@l when murine cells were cultured, and PHA-P was added at a ~ncen~ation of 1 pg/ml when human cells were cultured. As shown in Table II, the incorporation of [ 3H]thymidine into murine spleen ceils was signXcantly increased by 17% to 150% compared with the
TABLE I Effect of bestatin in the absence of mitogens on murine and human spleen cells and thymocytes
Bestatin concentration
Murine cells WC
Control 0 (~~ml)
1000 100 10 1 0.1
8425 f 6731 * 18?2&t 13379f 11399f 12376f
Human cells
-I 545 (100)” 969E ( 79.9) 595Sb (222.9) 2462b (158.8) 1904u(1353) 2178’ (146.9)
%Y
SPC
6837 & 604 (108) 6215 f 640 ( 90.9) 6816 it 1077 ( 99.7) 662.5t 682 ( 96.9) 7145 f 1179 (104.5) 6741 ?: 1199 ( 98.6)
126362 1857 + 55722 9932 + 11195f 12914 f
Thy 992 (100) 20$ ( 14.7) 117c ( 44.1) 596” ( 78.6) 291” ( 88.6) 852 (102.2)
9263 f 1954i 4456p 6336 f 7355 * 8485 k
996 (100) 27’ ( 21.1) 120’ ( 48.1) 380’ ( 68.4) 647’ ( 79.4) 1086 ( 91.6)
Cells were treated with various concentrations of bestatin for 72 h, then the [3H]thymidine incorporation assay was done. The values represent the mean f SD of three experiments carried out in quadruplicate; values in parentheses &e relative incorporation (%). s Calculated as (cpm in tested welt)/(cpm in control well) x 100 (X) b Si8nilkant increase compared to control (0 pg/ml of bestatin) by Stndent’s z-test (p < 0.05). E Significant decrease compared to control ( p < 0.05).
TABLE II Bffect of bestatin in the presence of mitogens on mu&e and human spleen cells and thymocytes Bestatin concentration
Control 0 (F&ml) 1000 too 10
1 0.1
Murine cells (Con A addedb)
Humancells (PITA-P added=)
SPC
Thy
SPC
Thy
30610 2 4662 (100)” 3918 & 129’ ( 12.8) 76647 f 16096d (250.4) 43282 + 3203d (141.4) 35844 f 4086“ (117.1) 39946 + 399Sd (138.5)
23052 f 1208 (100) 7169 f 222’ ( 31.1) 46081 it 7926d (199.9) 30936 f 43934 (134.2) 22061 rs 2008 f 95.7) 22752 i 1820 ( 98.7)
45420 f 1791 (loo) 7494 + 135” ( 16.5) 22574 f 406= ( 49.7) 41196 ?: 1565“ ( 90.7) 44739 f 761 ( 98.s) 45920 f 1561 (iOl.1)
34587 k 1607 (loo) 31405 + 1916’ ( 99.8) 31025 it. 127T ( 89.7) 30882 f 2653” ( 89.2) 34864 f 6206 (l~.S) 35555 f 4124 (1~2.8~
Cklls were treated with various eoncentrat~ns of bestatin with mitogens for 72 h, then the [sH~th~i~ne ~co~~tion assay was done. The values represent the mean + SD of three experiments carried out in qmtdruplicatc; values in parentheses pive relative incorporation (%). * Calcuiated as (cpm in tested well)/(cpm in control well) x 100 (%) b 5 flg/rnl of Con A was ad&d into each well. c 1 pg/ml of PHA-P was added into each well. * Significant increase compared to control (mitogen alone) by Student’s t-test (p c 0.05). o Significant decrease compared to control (p < 0.05).
control, Con A alone. Namely, be&&in stimulated mitogenicity of Con A on murine spleen cells. 1000 pg/ml of bestatin, a very high dose, may probably exert a ~~oto~c effect on spleen cells. Bestatin also signifkantly stimulated mitogenicity of Con A on murine thymocytes at 100 to
10 pgfml. In addition, by using 1 or 10 pg/ml of Con A, similar stimulator effects of bestatin on mutine cells were observed (data not shown in this paper). On the contr~y, mitogeni~ity of PI-IA-P on human spleen cells and thymocytes was inhibited by more than 10 pg/ml of bestatin
after 72 h treatment,
but bestatin less than 1 pg/ml, clinically used concentrations, showed no effect on mitogeniciry of PHA-P (Table II). Similar results were obtained in the case of 48 h treatment of bestatin in the presence of PHA-P, PWM or Con A (Fig. 1). In order to determine whether bestatin exerts its effect on only T lymphocytes or not, spleen cells from athymic nude mice were cultured with bestatin for 48 h or 72 h in the absence or presence of 5 p&/ml of LPS as a I? cell mitogen. As shown in Pig. 2, the incorporation of [ 3H]thymidine into athymic mouse spleen cells was significantly increased by 72 h treatment of 100 to 1000 pgjmi ofbestatin in the absence of LPS, and mitogenicity rf LPS was also enhanced by the combination treatment of 100 to lOOO&ml of bestatin compared with the LPS alone. Almost similar results were found after 48 h bestatin treatment (data not shown). These resuit may
0
loo00
I’H
zoo00
sooon
&mo
0
Fig. 2. Etkt of bcstatin in the absence or presence of LPS on athymic mouse spleen cells. Cells were treated for 72 h, then the [‘H]thymidine incorporation assay was done. L’ach column and bar represent the mean and SD of three exp& ments. ’ Significant increase compared to control (p < 0.05). b Significant increase compared to LPS alone (p c 0.05).
suggest that bestatin activated the DNA synthesis of murine B cells at a very high concentration, more than 100 pg/ml. Additional experiments by the same assay system were performed with human peripheral blood lymphocytes (PBL). As shown in Fig. 3, mitogenicity of PHA-P on human PBL was inhibited dose-dependently as in the case of human spleen cells, although no significant effect was observed by the treatment of bestatin alone. Eflecctof bestatin on non-aa!herent lymphocytes
Fig. I. Effect of bestath combined with PHA-P, PWM. Con A on human spleen cells. Cells were treated for 48 h, then the [‘Hlthymidine incorporation assay was done. Each column and bar represent the mean and SD of three expcriments. a Significant decrease compared to PHA-P alone by Student’s r-test (p < 0.05). ’ Significant decrease compared to PWM alone (p c 0.05). F Significant decrease compared to Can A alone (p c 0.05).
We examined the etkct of bestatin on three groups of murine spleen cells by the [ 3H]thymidine incorporation assay in order to elucidate whether the mechanism of its effect is related to the existence of adherent cells (macrophages and dendritic ceils). The cell population of each group was described in Materials and Methods. In this assay, bestatin was added at concentrations of 10, 100, 1000 pglml, and cells were treated for 48 h. As shown in Fig. 4, bestatin increased incorporation of [ 3H]thymidine at 10 to 1000 pg/ml in the absence of Con A in a!1 three groups. In the presence of 5 pgjml of Con A, bestatin also stimulated
168 mitogenicity of Con A in all groups at 100 to 1000 pgj’ml. These results suggested that bestatin
PtlAt3sl0 Pth3s1.0
Fig.3. Effect of bestatin in the absence or Presence of PHA-P on human peripheral blood lymphocytes. Cells were treated for 48 h, then the [‘H]thymidine incorporation assay was done. Each Golumnand bar represent the mean and SD of three ex~riments. a S~~j~t decrease compared to PHA-P alone by Student’s t-test ( p e 0.005).
Fig. 4. meet of bestatin on three groups of murine spieen cells. AUgroups were treated with various concentrations of bestatin in the absence or presence of Con A for 48 h, then the [“W]thymidineincorpordion assay was done. Each column and bar represent the mean and SD of three experiments.
showed the enhancing e%ct on the DNA synthesis of murine spleen cells by acting directly on T cells. Next, we examined the effect of bestatin on human non-adherent spleen celis and peripheral blood lymphocytes (PBL) by the same procedure as murine cases. In this assay, bestatin was added at concentrations of ~.l-lOOO~g~m1 in the absence or presence of PHA-P, Bestatin inhibited the DNA synthesis of human non-adherent spleen cells and also inhibited the PHA-induced mitogenicity of them (Fig. 5). Similarly, it inhibited the PHA-induced mitogenicity of human nonadherent PBLs (data not shown). E&et of best&in on DNA synthesis of human leukemic cells in vitro
The same assay was performed with HL-60, a human p~myeloc~ic leukemia cell line, and MOLT-3, a human T lymphoblastic leukemia cell
Fig. 5. Effect of bestatin in the absence or presence of PHA-P on human non-adherent spleen cells. Cekiswere treated for 48 h, then the [‘Hlthymidine incorporation assay was done. Each column and bar represent the mean and SD of three experiments. ” Significant decrease comp-ed to control (P-C 0.05). b Signihcanl decrease compared 10 PHA-P alone (p 4 OM).
I69 TABLE III Effect of bestatin on leukemic cells, HL-60 and MOLT-3 Bestatin concentration
Control 0 (&nl) loo0 100 10 I 0.1
HL-60
MOLT-3
72 h
96 h
loo;’ 0.8 f O.lb 14.7 f 4.9b 48.8 f 14.1’ 74.5 f 10.6’ 93.7 f 7.3’
IO0 1.5 f 10.8 f 29.7 f 67.2 + 91.1 +
0.4b 1.3b 2.4b 2.P 3.T
72 h
96 h
100 42.9 +_1.P 37.1 * 2.P 65.3 f 2.Sb 76.6 + 3.5’ 99.1 f 6.3
IO0 56.6 f 4.4b 28.3 + 2.3b 45.9 + 2.7b 83.4 +-4.6b 104.6 + 7.2
Cells were treated with various concentrations ofbestatin for 72 h or 96 h.then the [‘Hlthymidine incorporation assay was done. The values represent the mean k SD (relative %) for quadrupticares from one of three similar experiments. il Calculated as [cpm in tested well)/(cpm in control well) x 100 (%) ’ Significant decrease compared to control (0 &ml of best&n) by Student’s r-test (p c 0.005). c Significant decrease compared to control (p < 0.05).
line in order to compare with the effects of bestatin on lymphocytes as described above. As shown in Table III, bestatin showed inhibitory elects against the DNA synthesis of both HL-60 and MOLT-3 cells after 72 h or 96 h treatment. The growth inhibition of HL-60 cells by bestatin was observed within the wide range of concentrations of 0.1-1000 &ml in a dose dependent manner, while in MOLT-3, the inhibition was observed at the concentration of more than l.Opg/ml of bestatin. Discussion
We show in this paper that bestatin has a growth inhibitory effect on human lymphocytes contrary to growth stimulation on murine lymphocytes. There have been many reports about the immunoenhancing e&t of bestatin, especially that it has been shown to augment T cell mitogenesis. Saito et al. (1977, 1978) showed the stimulatory effect of best&n on the blastogenesis of guinea pig lymphocytes. Ishizuka et al. (1980) showed that bestatin increased [ 3H]thymidine incorporation into murine lymphocytes, and augmented the mitogenicity of Con A or LPS. These results were
consistent with our results in murine spleen cells. However, the effect of bestatin on human lymphocyte proliferation has not been well studied. Morikawa et al. (1989) reported the suppressive e&t of bestatin on the proliferation and differentiation of human B cells in vitro dose-dependently. Their results correlated to our results, but we show that bestatin inhibited the DNA synthesis of both human T and 3 cells. How does bestatin work as an inhibitor to human T cell proliferation and as a stimulator to murine T cell proliferation? Ishizuka et al. (1980) and Weiljmann et al. (1985) showed that bestatin worked to murine lymphocytes via macrophages. However, our results indicate that bcstatin worked directly to murine lymphocytes to enhance ceU growth and Con A reactivity. We also found that bestatin worked directly to human lymphocytes to inhibit cell growth and PHA-induced mitogenicity. Recently, several studies have reported on the cell surface aminopeptidase activities of both human and murine cells. Amoscato et al. (1989) demonstrated that leucine aminopeptidase-like activity existed on the membrane of human lymphocytes and was inhibited by bestatin. Look et al. (1989) and Ashmun et al. (1990) found that cell surface glycoprotein CDI3, which
was exxpressedan human mon~~es, granuloq&es and myeioid hmkemia ceh lines, was identical to ~o~~t~dase N, and that this enzyme activity was inhibited by bestatin. On the other hand Gorvel et al. ( 1990) and Vivier et al. ( 1941) demonstrated that mouse thymocyte-activating molecule (THAM) was associated with an aminopeptidase N activity and was the counterpart of the hnman CD26 (di~~t~dyi peptidase XV).From these recent report% it is suggested that surface ~~ope~~dase c&d play an ~rn~~~t role in cdt activation, but the relation between best&n’s ef&ct and ~~b~ti~n of ~ino~Fti~e is now m&r ~v~~~tion. We recaE the fact that bestatin had a direct growth ~n~b~to~ elect aa;ainst HL-69, MOLT-3 (in this paper and Sakurads et al., 1990) and choriocarcinoma cells (Ino et al,, 1991). It also works as an inhibitor oa epidermal growth factorinduced DNA synthesis in primary cultured rat hiatuses (Takahashi etal., 19g9). These results indicated that best&in had direct grow& inhibitory effect to some kinds of human cancer cdf lines and normal cells. In this paper; we show that best&n works as an ~~os~u~~or to mtine lyrn~h~~es, but not human ~~rnphocy~~ at higher dose than cl&ical use. We and others showed that best&n had growth inhiitory effects to human cancer cell lines. These results encourage us to use bestatin for cancer therapy. However, we have to check the immunosuppressive effect of this drug at high dose.
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