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Effects of levamisole on human natural killer and killer cell activity and production of interferons
Effects of Levamisole on Human Natural Killer and Killer Cell Activity and Production of Interferons Marina Liberati, Ernest C. Borden, Jack A. McBain, and Richard Raubertas Abstracl: The effect of levamisole on natural killer (NK) and killer (K) cell activities and on interferon production was determined in both normal subjects and cancer patients. Levamisole had no effect on these cytolytic immune effector cells in vitro. It did not induce interferon production. Sequential determinations of NK cell activity and interferon plasma levels after single oral doses of levamisole failed to demonstrate an effect of levamisole on spontaneous cytotoxicity or interferon production.
Key Words: Levamisole; Natural killercells; Interferons.
INTRODUCTION Levamisole has increased survival of mice with transplanted tumors, particularly when used in conjunction with chemotherapy (Chirigos et al., 1973). Clinical benefit of levamisole has been suggested from studies in advanced Ioco-regional breast carcinoma (Rojas el al., 1976), resectable lung carcinoma (Amery, 1978), multiple myeloma (Salmon et al.. 1982), and metastatic colorectal carcinoma (Borden et al., 1982a). The mechanism of antitumor action of levamisole remains incompletely defined. The drug increased monocyte function (Pike and Snyderman, 1976; Schmidt and Douglas, 1976; Nathanson et al.. 1978) and maturation of T cells (Renoux and Renoux, 1977; Van Wauwe and Van Nijen, 1977). Effects on other cytolytic immune effector cells have not been evaluated. To further investigate the mechanism of the antitumor activity of levamisole, we studied: I) its effects on natural killer (NK) cell function, killer (K) cell cytotoxicity, and interferon (IFN) production in vitro by lymphocytes from both healthy subjects and from cancer patients, and 2) the effect of levamisole administration on IFN induction and spontaneous cell-mediated cytotoxicity in vivo.
Received June 26, 1981; revised January 21, 1982. From the Division of Clinical Oncology, University of Wisconsin. Clinical Cancer Center and VA Hospitals, Madison, Wisconsin. Address requests for reprints to: Dr. Ernest C. Borden, Departments of Human Oncology and Medicine, University of Wisconsin, 600 Highland Avenue, Madison, WI 53792. © ElsevierSciencePublishingCo., inc., 1982 52 VanderbiltAve., New York. N.Y. Immunopharmacotogy5, 11-18 (1982)
11 0162-3109/82/0501108502.75
12
M. Liberati, et al.
MATERIALS AND METHODS
Lymphocyte Donors Heparinized blood was drawn from healthy donors varying from 18 to 40 years of age. Additional blood samples were obtained from six patients with advanced malignancies. The Ficoll-Hypaque method was used to isolate mononuclear cells (PMBC) from the blood. Cells recovered from the interface ring were washed three times with calcium- and magnesiumfree phosphate-buffered saline (PBS) and resuspended in RPMI 1640 medium (Grand Island Biological Co., Grand Island, NY) containing 24 mM N-2-hydroxy ethylipiperazene-N'-2ethanesulfonic acid (Hepes) buffer, gentamicin (50 pg/ml), and 10% fetal calf serum (FCS). This medium will be referred to as "complete" medium. Plastic adherent cells were depleted by incubating (in humidified air, 5% CO 2 at 37°C) 107 PMBC per ml in complete medium for I hr in Falcon plastic tissue culture flasks (surface area 25 cm 2) The percentage of monocytes before and after surface adherence was determined by nonspecific esterase staining.
Target Cells The human myeloid cell line K562 was used for detection of natural cytotoxicity. This cell line was maintained as a suspension culture in RPMI 1640 supplemented with 10% pooled human serum. Chang liver cells were used for antibody-mediated cytotoxicity test. These cells were grown as monolayers in Eagle's minimal essential medium (MEM; Grand Island Biological Co., Grand Island, NY) with 10% FCS.
Chemicals Levamisole was obtained from Janssen R & D Inc. (New Brunswick, NJ) as dry crystalline preparation and dissolved in complete medium to the desired concentrations. Polyinosinicpolycytidylic acid (poky l:poly C) was purchased from Miles Laboratory (Elkhart, IN) and dissolved in Dulbecco's phosphate-buffered solution (DPBS).
Chromium Release Assay From 2 to 5 × 100 target cells were labeled by incubation with i00 ~Ci of SICr as sodium chromate (Na~SICrO4; New England Nuclear, Boston. MA) in a 0.5 ml final volume of complete medium for 45 min (Chang cells) or 90 min (K562 cells) in a water bath at 37°C Labeled target cells were mixed in 96 well round-bottomed microplate (Linbro Scientific, Hamden, CT) with various concentrations of effector cells. Four replicate wells were used for each effector to target ratio. Rabbit anti-Chang cell serum (a gift of Steven Shore, Center for Disease Control, Atlanta, GA) was used to perform antibody-dependent cytotoxic assay (Shore et al., 1977). Mixtures of effector and target cells were incubated at 37°C for 5 hr to perform NK cell cytotoxic test, or for 8 hr when antibody-mediated lysis against Chang cells was tested. Supernatants were then collected, using a Titertek Supematant Harvesting System (Flow Laboratories, Rockville. MD). A medium control provided an estimate of spontaneous release of SICr from the target cells
Abbreviations. NK: natural killer; K: killer, IFN: interferon; PMBC: peripheral mononuclear cells: PBS: phosphate-buffered saline; Hepes: N-2-hydroxyethyl piperasme-N'-2-ethanesulfonic acid: FCS: fetal calf serum; MIFM:minimal essential medium; DPBS: Dulbecco's phosphate-buffered saline: poly I: poly C: polyinosinic-polycytidylicacid.
Levamisole: Effects on NK and K Cell Activity
13
(spontaneous cpm). Total SlCr release (total cpm) was determined by completely lysing the target cells with 0.1 ml of 5% cetramide. Percent specific lysis was computed using the formula:
% lysis =
test cpm - spontaneous cpm total cpm - spontaneous cpm
× 100
From 3 to 5 effector to target ratios were tested for each treatment group.
Statistical Analysis As mean specific 51Cr release increased, so also did the standard deviation of the mean. To correct this trend, a logarithmic transformation was performed prior to statistical analyses. For all quadruplicate sets of wells within an assay, interwell variability was pooled to calculate a pooled standard error. Differences in log-specific SlCr release were standardized (using the pooled standard error) and assessed for statistical significance by comparison to the distribution of studentized range statistics (Snedecor and Cochran, 1967). This allowed all mean log SlCr release values for an assay to be compared with each other, while maintaining a significance level of 0.05 for all tests taken together. Differences in the proportions of subjects who showed either increase or decrease of NK killing over a 24 hr period of observation between the group who received an oral dose of levamisole and the control group were tested using Fisher's exact test.
Interferon Assay Interferon titer was assayed via a quantitative colorimetric assay (Borden and Leonhardt, 1977). Dilutions of each PMBC culture supernatant or plasma sample were added in duplicate to confluent BG9 human foreskin fibroblast monolayers. After 24 hr at 37°C the cells were infected with vesicular stomatitis virus. Human leukocyte and fibroblast interferon laboratory standards were incorporated into each assay. They had been repeatedly titered against their respective international standards (IF ct and 13 obtained from the NIAID) to establish their relative tilers.
RESULTS The effect of levamisole on the lytic phase of K562 killing by PMBC from 7 healthy donors was examined at levamisole concentrations ranging from 0.005 to 50 p,g/ml (Figure i). Different lymphocyte donors had a wide range of spontaneous NK activity against the human tumor cell line K562 (Lucero et at., 1981; Borden et al., 1982b). Levamisole did not augment NK cell cytotoxicity in subjects with low, normal, or high basal NK reactivity (Figure 1). The effect of levamisole pretreatment on effector cells before addition to target cells and on IFN induction was also investigated. PMBC ( 8 - 1 0 × 106 in I0 ml of complete medium) were exposed for 18 hr to various drug concentrations (0.005-100/~g/ml). Effector cells from 5 normal donors and 6 cancer patients were assayed in 12 individual cytoloxicity tests. Overnight pretreatment of PMBC with levamisole also did not result in significant augmentation of cytotoxicity against K562 cells. The drug did not enhance depressed NK cell activity, which was observed in 2/6 patients studied. Interferon was not detected in any supernatants from the nine experiments in which it was assayed. To determine whether macrophages could be suppressing the effect of levamisole on cell cytotoxicity, adherent cells were removed from mononuclear cell populations before treatment with levamisole. Four different healthy donors in six individual assays were tested. Macrophage depletion (decrease of esterase-positive cells from 30 to 15% and from 17 to 5%) increased NK cell baseline activity against K562. However, no further enhancement of cytotoxicity resulted from treatment of effector cells with levamisole for 18 hr.
M. Liberati, et al.
14
5CC-K562
TRROETS
80. 70.l--(.b
60.50.-
#=..=4
Z CE) I-CD I-U
40.30.20.I0.O.
I 0
I .005
I .05
I .5
I 5
I 50
CONCENTRAT 18N[ UGIML ) Figure 1 Effect of a short treatment period (5 hr) with 0.005 to 50 I.u31ml levamisole on NK expression of 7 healthy subjects. The percent specific 51Cr release from labeled K562 target cells (+-SE) is shown for 50:1 effector to target ratio. To study levamisole's effect on the lytic phase of spontaneous cytotoxicity the drug (from 0.005 to 50 p.g/ml) was added to a mixture of effector and target cells and the mixtures were incubated for 5 hr. Supernatants were then collected and % 51Cr specific release was determined. Each symbol represents a different donor.
Experiments (PMBC from 3 different healthy donors were used) were undertaken to test the effect on NK cell function of levamisole in combination with suboptimal concentration of a known stimulant of NK cell activity. Poly I: poly C, at a previously defined optimal (100/J,g/ml) concentration (Borden et al.,1982b)enhanced NK cell reactivity against Chang and K562 target cells (Table I). Levamisole did not increase NK cell activity induced by suboptimal concentration (10 p,g/ml) of poly I: poly C (Table I). Other tested interval concentrations of levamisole (0.05-5/~g/ml) also resulted in no augmentation of poly I: poly C-induced NK cell cytotoxicity. Supematants from PMBC cultures treated with 10/J,g,/ml of poly I: poly C contained 1 0 0 - 1 5 0 IU of IFN Levamisole (from 0.05 to 10 p,g/ml) did not increase the yield of IFN by PMBC exposed to the suboptimal dose of PIC. Antibody-mediated cytotoxicity against Chang target cells of PMBC exposed to narrow (0.05-1/~g/ml) and wide (0.01 - I0/~g/ml) intervals of levamisole concentrations for short and long periods was investigated in six individual experiments. Neither exposure of effector cell to levamisole during the lytic phase of SICr release assay nor pretreatment of PMBC with the drug for 18 hr, 3 or 5 days resulted in a significant alteration of K cell cytotoxicity against Chang target cells (data not presented).
15
Levamisole: Effects o n NK and K Cell Activity
Table I
NK cell activity of human peripheral mononuclear cells treated with a combination of levamisole and poly I : poly C ° Spontaneous cell-mediated cytotoxicity (% 51Cr Release +- SE)
lmmunomodulator ( l~/ml) Poly I:poly C
Levamisole
0 I0 I0 10 I0
0 0 0. I I 10
I00
0
Chang 8.2 12.7 6.9 12.5 9.8
_+ 1.2 ± 1.5 ± 1.2 ± 1.5 ± 1.3
31.5 _+ 2.4 b
K562 45.3 55.0 52.2 50.1 46.2
± 3.2 _+ 3.8 ± 3.6 _+ 3.5 _+ 3.3
63.0 +_. 4.2 b
aPMBC were cultured for 6 hr with or without levamisole, 10 or i(30 p,g/ml of poly I: poly C were added and the cells incubated for an additional 12 hr. The cells were then harvested, washed once, resuspendecl in complete medium and diluted to the desired concentrations and tested for NK activity. Chang cell targets at effector:target cell ratio of I00: I and K562 target cells at effector:target cell ratio of I00: I. Results at lower effector:target ratios (50: I and 25: I) were idenlical. bp <0.05 compared to untreated.
The effect of in vivo administration of a single oral dose levamisole (2.5 mg/kg) on NK function was tested in 21 normal individuals. Sequential evaluation of plasma IFN levels and NK activity before (at 0 hr) and at 2, 4, and 24 hr after drug administration were performed. An additional blood sample at 48 hr was obtained for the IFN activity determination. PMBC and plasma samples were also obtained from 19 individuals not ingesting levamisole. K562 ceils were used as target in s p o n t a n e o u s cytotoxic tests. In both subjects receiving levamisole a n d in controls, NK cell cytotoxicity varied during a 24 hr period. However, no significant differences occurred b e t w e e n the control and levamisole groups in the frequency of NK cell stimulation (p > 0.7 for all the comparisons at 50: I and i 0 0 : I effector to target ratios by Fisher's exact test). The magnitude of NK cell activity variations was similar in the two groups (Table 2). Detectable levels of IFN were not found in any of the pretreatment plasma samples tested. The administration of an oral d o s e of levamisole did not result in a measurable IFN production.
Table 2
NK Cell Activity of Human Peripheral Mononuclear Cells in Subjects Receiving a Single Oral Dose of Levamisole and Controls a Cytolysis of K562 target cells Levamisole group (21)b Control group (I0)
0 hr
27.4 ~- 2.7c
26.7 "- 5.2
2 hr
30.5 -+ 3.3
28.6 - 5.4
4 hr
29.6 +- 2.8
30.0 -+ 6.5
24 hr
27.1 _+ 3.6
23.9 -+ 5.1
aPMBC were obtained before (at 0 hr), 2.4, and 24 hr after drug administration and tested in a single 51Cr release assay. Effector cells from subjects receiving levamisole and controls were assayed in parallel. bNumbers in parentheses indicate the number of subjects tested in each group at all 4 study points. In 9 additional controls, data were only obtained at some of the indicated time points. Although they are thus not pan of the formal analysis, their inclusion did not change the results. CMean of % SICr specific release ± SE at a 50:1 effector to target cell ratio.
16
M. Liberati, et al.
DISCUSSION Effector cell populations potentially important in tumor immunity include T cells, NK cells, K cells, and macrophages. Levamisole enhanced T-cell proliferation after stimulation with either mitogens (Hadden eta]., 1975; Lichtenfe]d et al., 1976; Al-lbrahim et al., 1977; Lewinshi et al., 1980) or antigens (Pabst and Crawford, 1975; Lichtenfeld eta]., 1976; A]-Ibrahim et al., 1977; Lewinshi et al., 1980). A rise in the number of both "active" (Wybran and Govaerts, 1977) and "total" (Ramot eta]., 1976; Wybran and Govaerts, 1977) T-cell forming E rosettes occurred both in vitro (Ramot eta]., 1976) and in vivo (Wybran and Govaerts, 1977; Ramot et al., 1976). Chemotaxis (Pike and Snyderman, 1976; Schmidt and Douglas, 1976; Nathanson eta]., 1978), phagocytosis (Schmidt and Douglas, 1976), and cytoplasmic spreading (Schmidt and Douglas, 1976) of human monocytes were increased by levamiso]e. In our study, levamisole had no effect on NK or K cell activity in vitro or in vivo. Thus, the antihumor effects of levamisole are most likely mediated by other mechanisms. Cell-mediated immune functions have been enhanced by interferons. Cytotoxicity of T lymphocytes (Heron et al., 1976; Zarling et al., 1978), NK (Herberman et at., 1979; Zarling et al., 1979), and K (Herberman et al., 1978) cells are augmented. Two different mechanisms are involved in the IFN boosting of spontaneous cytotoxicity: maturation of functionally active NK cells from inactive precursors (Herberman et a]., 1979; Targan and Dorey, 1980), and enhancement of lytic function of active NK cells (Targan and Dorey, 1980). Several observations suggested to us the possibility that, in some instances, IFN may mediate the immunological effects of levamisole: I) levamisole has been reported to affect the IFN production in mice (Matsubara et al., 1979); 2) both levamisole and [FN modulate the activity of T lymphocytes (Hadden et al., 1975; Pabst and Crawford, 1975; Heron et al.. 1976; Lichtenfeld et al., 1976; Ramot et al., 1976; Al-[brahim et al., 1977; Wybran and Govaerts, 1977; Zading el a]., 1978; Lewinshi et al., 1980); and 3) both these agents share the ability to regulate the maturative process of cells of T lineage (Renoux and Renoux, 1977; Van Wauwe and Van Nijen, 1977; Herberman et al., 1979; Targan and Dorey, 1980). However, we found no IFN either in vitro or in vivo after treatment with ]evamisole. Furthermore, we found no synergistic effect of IFN production in vitro of levamiso]e and a suboptimal dose of a potent IFN inducer. The absence of detectable IFN after in vitro treatment of PMBC with various concentrations of levamisole or in vivo following a single drug dose administration correlated with the lack of any effects (of the drug) on NK and K cell killing. Julie Ruff made important technical contributions to the initiation of this project. Supported by funds from the National Cancer Institute (CA 20432), the Veterans Administralion, and the Ortho Pharmaceutical Corporation. M. L. is a visiting scholar from the University of Perugia, Italy, supported by Lega ltaliana per la lotta controi tumori.
REFERENCES Al-lbrahim MS, Holzman RS, Lawrence HS (1977) Concentrations of levamisole required for enhanced proliferation of human lymphocytes and phagocytosis by macrophages. J Infect Dis 135:517. Amery WK (1978) Final results of a multicenter placebo-controlled ]evamisole study of resectable lung cancer. Cancer Treat Rep 62:1677. Borden EC, Leonhardt PH (1977) A quantitative semimicro, semiautomated colorimetric assay for interferon. J Lab Clin Med 89:1036. Borden EC, Davis TE, Crowley JJ, Wolberg WH, McKnight B, Chirigos MA (1982a) Interim
Levamisole: Effects on NK and K Cell Activity
17
analysis of a trial of levamisole and 5-fluorouracil in metastatic colorectal carcinoma. In Clinical Immunotherapy--1980. Eds., W Terry, S Rosenberg. New York: American Elsevier, in press. Borden EC, Hawkins MJ, Edwards BS, Liberati AM, Cantell K (1982b)Pharmacologic and immunologic effects of human leukocyte interferon in patients with neoplasia: an interim report. Proceedings: Conference on Clinical Potentials of Interferons in Viral Disease and Malignant Tumors. Ed., R Kono. Tokyo: Japan Medical Research Foundation, Uni,Jersity of Tokyo Press, pp. 169-179. Chirigos MA, Pearson JW. Pryor J (1973) Augmentation of chemotherapeutically induced remission of a murine leukemia by a chemical immunoadjuvant. Cancer Res 33:2615. Hadden, JW, Coffey RG, Hadden EM, Lopez-Corrales E, Sunshine GH (1975) Effects of levamisole and imidazole on lymphocyte proliferation and cyclic nucleotide levels. Cell Immunol 20:98. Herberman RB, Djeu JY, Ortaldo JR, Holden HT, West WH, Bonnard GD (1978) Role of interferon in augmentation of natural and antibody-dependent cell-mediated cytotoxicity. Cancer Treat Rep 62:1893. Herberman RB, Djeu JY, Kay HD, Ortaldo JR, Riccardi C, Bonnard GD, Holden HT. Fagnani R. Santoni A, Puccetti P (1979) Natural killer cells: characteristics and regulation of activity. Immunol Rev 44:43. Heron I, Berg K, Cantell K (1976) Regulatory effect of interferon on T cells in vitro. J Immunol 117:1370. Lewinski UH, Mavligit GM, Hersh EM (1980) Cellular immune modulation after a single high dose of levamisole in patients with carcinoma. Cancer 46:2185. Lichtenfeld J, Desner MR, Wiernik PH, Mardiney MR Jr (1976) Modulating effects of levamisole (NSC-177023) on human lymphocyte response in vitro. Cancer Treat Rep 60:571. Lucero MA, Fridman WH, Provost MA. Billardon C, Pouillart P, Dumont J, Falcoff E (1981) Effect of various interferons on the spontaneous cytotoxicity exerted by lymphocytes from normal and tumor-bearing patients. Cancer Res 41:294. Matsubara S. Suzuki F, Ishida N (1979) The induction of interferon by levamisole in mice. Cell Immunol 43:214. Nathanson SD. Zamfirescu PL, Portaro JK, deKernion JB, Fahey JL (1978) Acute effects of orally administered levamisole on random monocyte motility and chemotaxis in man. J Natl Cancer Inst 61:301. Pabst HF, Crawford J (1975) L-Tetramisole enhancement of human lymphocyte response to antigen. Clin Exp Immunol 21:468. Pike MC. Snyderman R (1976) Augmentation of human monocyte chemotactic response by levamisole. Nature 261:136. Ramot B. Biniaminov M, Shoham C, Rosenthal E (1976) Effect of levamisole on E-rosetteforming cells in vivo and in vitro in Hodgkin's disease. N Engl J IVied 294:809. Renoux G, Renoux M (1977) Thymus-like activities of sulphur derivatives on T-cell differentiation. J Exp Med 145:466. Rojas AF. Feierstein JN, Mickiewicz E, Glait H, Olivari AJ (1976) Levamisole in advanced human breast cancer. Lancet 1:21 I. Salmon SE, A]exanian R. Dixon D(1982) Chemoimmunotherapy for multiple mye]oma: effect of ]evamisole during maintenance. In Clinical Immunotherapy--1980. Eds., W Terry, S Rosenberg. New York: American Elsevier, in press. Schmidt ME. Douglas SD (1976) Effects of levamiso]e on human monocyte function and immunoprotein receptors. Clin Immunol Immunopathol 6:299.
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Shore SL, Melewicz M, Gordon DS (1977) The mononuclear cell in human blood which mediates antibody-dependent cellular cytotoxicity to virus-infected target cells. I. Identification of the population of effector cells. J Immunol 118:558. Snedecor GW, Cochran WG (1976) Statistical Methods. 6th e d Ames, IA: Iowa State University Press, 272. Targan S, Dorey F (1980) Interferon activation of "pre-spontaneous killer" (Pre-SK) cells and alteration in kinetics of lysis of both "Pre-SK" and active SK cells. J Immunol 124:2157. Van Wauwe J, Van Nijen G (1977) Effect of levamisole on autologous rosette-forming cells in nude mice. Clin Exp Immunol 30:465. Wybran J, Govaerts A (1977) Levamisole and human lymphocyte surface markers. Clin Exp Immunol 27:319. Zarling JM, Sosman J, Eskra L, Borden EC. Horoszewicz JS, Carter WA (1978) Enhancement of T cell cytotoxic responses by purified human fibroblast interferon. J Immunol 121:2002. Zarling JM, Eskra L. Borden EC, Horoszewicz J, Carter WA (1979) Activation of human natural killer cells cytotoxic for human leukemia cells by purified interferon. J Immunol 123:63.
Key Words: Levamisole; Natural killercells; Interferons.
INTRODUCTION Levamisole has increased survival of mice with transplanted tumors, particularly when used in conjunction with chemotherapy (Chirigos et al., 1973). Clinical benefit of levamisole has been suggested from studies in advanced Ioco-regional breast carcinoma (Rojas el al., 1976), resectable lung carcinoma (Amery, 1978), multiple myeloma (Salmon et al.. 1982), and metastatic colorectal carcinoma (Borden et al., 1982a). The mechanism of antitumor action of levamisole remains incompletely defined. The drug increased monocyte function (Pike and Snyderman, 1976; Schmidt and Douglas, 1976; Nathanson et al.. 1978) and maturation of T cells (Renoux and Renoux, 1977; Van Wauwe and Van Nijen, 1977). Effects on other cytolytic immune effector cells have not been evaluated. To further investigate the mechanism of the antitumor activity of levamisole, we studied: I) its effects on natural killer (NK) cell function, killer (K) cell cytotoxicity, and interferon (IFN) production in vitro by lymphocytes from both healthy subjects and from cancer patients, and 2) the effect of levamisole administration on IFN induction and spontaneous cell-mediated cytotoxicity in vivo.
Received June 26, 1981; revised January 21, 1982. From the Division of Clinical Oncology, University of Wisconsin. Clinical Cancer Center and VA Hospitals, Madison, Wisconsin. Address requests for reprints to: Dr. Ernest C. Borden, Departments of Human Oncology and Medicine, University of Wisconsin, 600 Highland Avenue, Madison, WI 53792. © ElsevierSciencePublishingCo., inc., 1982 52 VanderbiltAve., New York. N.Y. Immunopharmacotogy5, 11-18 (1982)
11 0162-3109/82/0501108502.75
12
M. Liberati, et al.
MATERIALS AND METHODS
Lymphocyte Donors Heparinized blood was drawn from healthy donors varying from 18 to 40 years of age. Additional blood samples were obtained from six patients with advanced malignancies. The Ficoll-Hypaque method was used to isolate mononuclear cells (PMBC) from the blood. Cells recovered from the interface ring were washed three times with calcium- and magnesiumfree phosphate-buffered saline (PBS) and resuspended in RPMI 1640 medium (Grand Island Biological Co., Grand Island, NY) containing 24 mM N-2-hydroxy ethylipiperazene-N'-2ethanesulfonic acid (Hepes) buffer, gentamicin (50 pg/ml), and 10% fetal calf serum (FCS). This medium will be referred to as "complete" medium. Plastic adherent cells were depleted by incubating (in humidified air, 5% CO 2 at 37°C) 107 PMBC per ml in complete medium for I hr in Falcon plastic tissue culture flasks (surface area 25 cm 2) The percentage of monocytes before and after surface adherence was determined by nonspecific esterase staining.
Target Cells The human myeloid cell line K562 was used for detection of natural cytotoxicity. This cell line was maintained as a suspension culture in RPMI 1640 supplemented with 10% pooled human serum. Chang liver cells were used for antibody-mediated cytotoxicity test. These cells were grown as monolayers in Eagle's minimal essential medium (MEM; Grand Island Biological Co., Grand Island, NY) with 10% FCS.
Chemicals Levamisole was obtained from Janssen R & D Inc. (New Brunswick, NJ) as dry crystalline preparation and dissolved in complete medium to the desired concentrations. Polyinosinicpolycytidylic acid (poky l:poly C) was purchased from Miles Laboratory (Elkhart, IN) and dissolved in Dulbecco's phosphate-buffered solution (DPBS).
Chromium Release Assay From 2 to 5 × 100 target cells were labeled by incubation with i00 ~Ci of SICr as sodium chromate (Na~SICrO4; New England Nuclear, Boston. MA) in a 0.5 ml final volume of complete medium for 45 min (Chang cells) or 90 min (K562 cells) in a water bath at 37°C Labeled target cells were mixed in 96 well round-bottomed microplate (Linbro Scientific, Hamden, CT) with various concentrations of effector cells. Four replicate wells were used for each effector to target ratio. Rabbit anti-Chang cell serum (a gift of Steven Shore, Center for Disease Control, Atlanta, GA) was used to perform antibody-dependent cytotoxic assay (Shore et al., 1977). Mixtures of effector and target cells were incubated at 37°C for 5 hr to perform NK cell cytotoxic test, or for 8 hr when antibody-mediated lysis against Chang cells was tested. Supernatants were then collected, using a Titertek Supematant Harvesting System (Flow Laboratories, Rockville. MD). A medium control provided an estimate of spontaneous release of SICr from the target cells
Abbreviations. NK: natural killer; K: killer, IFN: interferon; PMBC: peripheral mononuclear cells: PBS: phosphate-buffered saline; Hepes: N-2-hydroxyethyl piperasme-N'-2-ethanesulfonic acid: FCS: fetal calf serum; MIFM:minimal essential medium; DPBS: Dulbecco's phosphate-buffered saline: poly I: poly C: polyinosinic-polycytidylicacid.
Levamisole: Effects on NK and K Cell Activity
13
(spontaneous cpm). Total SlCr release (total cpm) was determined by completely lysing the target cells with 0.1 ml of 5% cetramide. Percent specific lysis was computed using the formula:
% lysis =
test cpm - spontaneous cpm total cpm - spontaneous cpm
× 100
From 3 to 5 effector to target ratios were tested for each treatment group.
Statistical Analysis As mean specific 51Cr release increased, so also did the standard deviation of the mean. To correct this trend, a logarithmic transformation was performed prior to statistical analyses. For all quadruplicate sets of wells within an assay, interwell variability was pooled to calculate a pooled standard error. Differences in log-specific SlCr release were standardized (using the pooled standard error) and assessed for statistical significance by comparison to the distribution of studentized range statistics (Snedecor and Cochran, 1967). This allowed all mean log SlCr release values for an assay to be compared with each other, while maintaining a significance level of 0.05 for all tests taken together. Differences in the proportions of subjects who showed either increase or decrease of NK killing over a 24 hr period of observation between the group who received an oral dose of levamisole and the control group were tested using Fisher's exact test.
Interferon Assay Interferon titer was assayed via a quantitative colorimetric assay (Borden and Leonhardt, 1977). Dilutions of each PMBC culture supernatant or plasma sample were added in duplicate to confluent BG9 human foreskin fibroblast monolayers. After 24 hr at 37°C the cells were infected with vesicular stomatitis virus. Human leukocyte and fibroblast interferon laboratory standards were incorporated into each assay. They had been repeatedly titered against their respective international standards (IF ct and 13 obtained from the NIAID) to establish their relative tilers.
RESULTS The effect of levamisole on the lytic phase of K562 killing by PMBC from 7 healthy donors was examined at levamisole concentrations ranging from 0.005 to 50 p,g/ml (Figure i). Different lymphocyte donors had a wide range of spontaneous NK activity against the human tumor cell line K562 (Lucero et at., 1981; Borden et al., 1982b). Levamisole did not augment NK cell cytotoxicity in subjects with low, normal, or high basal NK reactivity (Figure 1). The effect of levamisole pretreatment on effector cells before addition to target cells and on IFN induction was also investigated. PMBC ( 8 - 1 0 × 106 in I0 ml of complete medium) were exposed for 18 hr to various drug concentrations (0.005-100/~g/ml). Effector cells from 5 normal donors and 6 cancer patients were assayed in 12 individual cytoloxicity tests. Overnight pretreatment of PMBC with levamisole also did not result in significant augmentation of cytotoxicity against K562 cells. The drug did not enhance depressed NK cell activity, which was observed in 2/6 patients studied. Interferon was not detected in any supernatants from the nine experiments in which it was assayed. To determine whether macrophages could be suppressing the effect of levamisole on cell cytotoxicity, adherent cells were removed from mononuclear cell populations before treatment with levamisole. Four different healthy donors in six individual assays were tested. Macrophage depletion (decrease of esterase-positive cells from 30 to 15% and from 17 to 5%) increased NK cell baseline activity against K562. However, no further enhancement of cytotoxicity resulted from treatment of effector cells with levamisole for 18 hr.
M. Liberati, et al.
14
5CC-K562
TRROETS
80. 70.l--(.b
60.50.-
#=..=4
Z CE) I-CD I-U
40.30.20.I0.O.
I 0
I .005
I .05
I .5
I 5
I 50
CONCENTRAT 18N[ UGIML ) Figure 1 Effect of a short treatment period (5 hr) with 0.005 to 50 I.u31ml levamisole on NK expression of 7 healthy subjects. The percent specific 51Cr release from labeled K562 target cells (+-SE) is shown for 50:1 effector to target ratio. To study levamisole's effect on the lytic phase of spontaneous cytotoxicity the drug (from 0.005 to 50 p.g/ml) was added to a mixture of effector and target cells and the mixtures were incubated for 5 hr. Supernatants were then collected and % 51Cr specific release was determined. Each symbol represents a different donor.
Experiments (PMBC from 3 different healthy donors were used) were undertaken to test the effect on NK cell function of levamisole in combination with suboptimal concentration of a known stimulant of NK cell activity. Poly I: poly C, at a previously defined optimal (100/J,g/ml) concentration (Borden et al.,1982b)enhanced NK cell reactivity against Chang and K562 target cells (Table I). Levamisole did not increase NK cell activity induced by suboptimal concentration (10 p,g/ml) of poly I: poly C (Table I). Other tested interval concentrations of levamisole (0.05-5/~g/ml) also resulted in no augmentation of poly I: poly C-induced NK cell cytotoxicity. Supematants from PMBC cultures treated with 10/J,g,/ml of poly I: poly C contained 1 0 0 - 1 5 0 IU of IFN Levamisole (from 0.05 to 10 p,g/ml) did not increase the yield of IFN by PMBC exposed to the suboptimal dose of PIC. Antibody-mediated cytotoxicity against Chang target cells of PMBC exposed to narrow (0.05-1/~g/ml) and wide (0.01 - I0/~g/ml) intervals of levamisole concentrations for short and long periods was investigated in six individual experiments. Neither exposure of effector cell to levamisole during the lytic phase of SICr release assay nor pretreatment of PMBC with the drug for 18 hr, 3 or 5 days resulted in a significant alteration of K cell cytotoxicity against Chang target cells (data not presented).
15
Levamisole: Effects o n NK and K Cell Activity
Table I
NK cell activity of human peripheral mononuclear cells treated with a combination of levamisole and poly I : poly C ° Spontaneous cell-mediated cytotoxicity (% 51Cr Release +- SE)
lmmunomodulator ( l~/ml) Poly I:poly C
Levamisole
0 I0 I0 10 I0
0 0 0. I I 10
I00
0
Chang 8.2 12.7 6.9 12.5 9.8
_+ 1.2 ± 1.5 ± 1.2 ± 1.5 ± 1.3
31.5 _+ 2.4 b
K562 45.3 55.0 52.2 50.1 46.2
± 3.2 _+ 3.8 ± 3.6 _+ 3.5 _+ 3.3
63.0 +_. 4.2 b
aPMBC were cultured for 6 hr with or without levamisole, 10 or i(30 p,g/ml of poly I: poly C were added and the cells incubated for an additional 12 hr. The cells were then harvested, washed once, resuspendecl in complete medium and diluted to the desired concentrations and tested for NK activity. Chang cell targets at effector:target cell ratio of I00: I and K562 target cells at effector:target cell ratio of I00: I. Results at lower effector:target ratios (50: I and 25: I) were idenlical. bp <0.05 compared to untreated.
The effect of in vivo administration of a single oral dose levamisole (2.5 mg/kg) on NK function was tested in 21 normal individuals. Sequential evaluation of plasma IFN levels and NK activity before (at 0 hr) and at 2, 4, and 24 hr after drug administration were performed. An additional blood sample at 48 hr was obtained for the IFN activity determination. PMBC and plasma samples were also obtained from 19 individuals not ingesting levamisole. K562 ceils were used as target in s p o n t a n e o u s cytotoxic tests. In both subjects receiving levamisole a n d in controls, NK cell cytotoxicity varied during a 24 hr period. However, no significant differences occurred b e t w e e n the control and levamisole groups in the frequency of NK cell stimulation (p > 0.7 for all the comparisons at 50: I and i 0 0 : I effector to target ratios by Fisher's exact test). The magnitude of NK cell activity variations was similar in the two groups (Table 2). Detectable levels of IFN were not found in any of the pretreatment plasma samples tested. The administration of an oral d o s e of levamisole did not result in a measurable IFN production.
Table 2
NK Cell Activity of Human Peripheral Mononuclear Cells in Subjects Receiving a Single Oral Dose of Levamisole and Controls a Cytolysis of K562 target cells Levamisole group (21)b Control group (I0)
0 hr
27.4 ~- 2.7c
26.7 "- 5.2
2 hr
30.5 -+ 3.3
28.6 - 5.4
4 hr
29.6 +- 2.8
30.0 -+ 6.5
24 hr
27.1 _+ 3.6
23.9 -+ 5.1
aPMBC were obtained before (at 0 hr), 2.4, and 24 hr after drug administration and tested in a single 51Cr release assay. Effector cells from subjects receiving levamisole and controls were assayed in parallel. bNumbers in parentheses indicate the number of subjects tested in each group at all 4 study points. In 9 additional controls, data were only obtained at some of the indicated time points. Although they are thus not pan of the formal analysis, their inclusion did not change the results. CMean of % SICr specific release ± SE at a 50:1 effector to target cell ratio.
16
M. Liberati, et al.
DISCUSSION Effector cell populations potentially important in tumor immunity include T cells, NK cells, K cells, and macrophages. Levamisole enhanced T-cell proliferation after stimulation with either mitogens (Hadden eta]., 1975; Lichtenfe]d et al., 1976; Al-lbrahim et al., 1977; Lewinshi et al., 1980) or antigens (Pabst and Crawford, 1975; Lichtenfeld eta]., 1976; A]-Ibrahim et al., 1977; Lewinshi et al., 1980). A rise in the number of both "active" (Wybran and Govaerts, 1977) and "total" (Ramot eta]., 1976; Wybran and Govaerts, 1977) T-cell forming E rosettes occurred both in vitro (Ramot eta]., 1976) and in vivo (Wybran and Govaerts, 1977; Ramot et al., 1976). Chemotaxis (Pike and Snyderman, 1976; Schmidt and Douglas, 1976; Nathanson eta]., 1978), phagocytosis (Schmidt and Douglas, 1976), and cytoplasmic spreading (Schmidt and Douglas, 1976) of human monocytes were increased by levamiso]e. In our study, levamisole had no effect on NK or K cell activity in vitro or in vivo. Thus, the antihumor effects of levamisole are most likely mediated by other mechanisms. Cell-mediated immune functions have been enhanced by interferons. Cytotoxicity of T lymphocytes (Heron et al., 1976; Zarling et al., 1978), NK (Herberman et at., 1979; Zarling et al., 1979), and K (Herberman et al., 1978) cells are augmented. Two different mechanisms are involved in the IFN boosting of spontaneous cytotoxicity: maturation of functionally active NK cells from inactive precursors (Herberman et a]., 1979; Targan and Dorey, 1980), and enhancement of lytic function of active NK cells (Targan and Dorey, 1980). Several observations suggested to us the possibility that, in some instances, IFN may mediate the immunological effects of levamisole: I) levamisole has been reported to affect the IFN production in mice (Matsubara et al., 1979); 2) both levamisole and [FN modulate the activity of T lymphocytes (Hadden et al., 1975; Pabst and Crawford, 1975; Heron et al.. 1976; Lichtenfeld et al., 1976; Ramot et al., 1976; Al-[brahim et al., 1977; Wybran and Govaerts, 1977; Zading el a]., 1978; Lewinshi et al., 1980); and 3) both these agents share the ability to regulate the maturative process of cells of T lineage (Renoux and Renoux, 1977; Van Wauwe and Van Nijen, 1977; Herberman et al., 1979; Targan and Dorey, 1980). However, we found no IFN either in vitro or in vivo after treatment with ]evamisole. Furthermore, we found no synergistic effect of IFN production in vitro of levamiso]e and a suboptimal dose of a potent IFN inducer. The absence of detectable IFN after in vitro treatment of PMBC with various concentrations of levamisole or in vivo following a single drug dose administration correlated with the lack of any effects (of the drug) on NK and K cell killing. Julie Ruff made important technical contributions to the initiation of this project. Supported by funds from the National Cancer Institute (CA 20432), the Veterans Administralion, and the Ortho Pharmaceutical Corporation. M. L. is a visiting scholar from the University of Perugia, Italy, supported by Lega ltaliana per la lotta controi tumori.
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Levamisole: Effects on NK and K Cell Activity
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