Journal oflmmunolherapy 10:297-306 © 1991 Raven Press, Ltd., New York
Immunological Effects of Levamisole In Vitro Joan H. Schiller, *Mary Lindstrom, Patricia L. Witt, Jacquelyn A. Hank, tDavid Mahvi, Randall J. Wagner, Paul Sondel, and Ernest C. Borden Departments of Human Oncology, *Biostatistics, and tSurgery, William S. Middleton Veterans Administration Hospital and University of Wisconsin Clinical Cancer Center, Madison, Wisconsin, U.S.A.
Summary: Levamisole, an antihelminthic drug with immunological properties, has recently been reported to have antitumor activity when administered with 5-fluorouracil in patients with Duke's C colorectal carcinoma. The mechanism of this antitumor effect is unknown, but has been postulated to be related to levamisole's immunomodulatory properties. To define further the immunomodulatory activities of levamisole, we studied the in vitro effects of levamisole on monocyte and lymphocyte cytotoxicity, activation, and proliferation; induction of cytokine-induced proteins; and expression of tumor-associated antigens. Experiments utilized peripheral blood mononuclear cells from normal donors incubated in the presence of increasing concentrations of levamisole (0.1 to 100 (j-g/ml). Levamisole had no consistent effect on induction of 2',5'-oligoadenylate synthetase activity or indoleamine-2,3-dioxygenase activity, or production of tumor necrosis factor. Levamisole had no effect on monocyte cytotoxicity or expression of HLA-DR, HLA-DQ, HLA-DP, and the Fc receptor. Similarly, levamisole had no significant effect on NK or LAK cytotoxicity or the immunological activation of T-lymphocytes, assessed by expression of CD3, CD4, CD8, CD16, CD25, and CD56. Proliferation of lymphocytes from normal donors, patients with benign polyps, and patients with malignancies, with or without IL-2 or irradiated LS174T cells, was not significantly increased overall. No significant enhancement in the expression of three tumor-associated antigens (880364, NRCO-4, and ING-1) and the intercellular adhesion molecule-1 (ICAM-1) antigen on four human cancer cell lines was observed following in vitro exposure to levamisole. We conclude that levamisole is not a potent modulator of the immune parameters we examined, and that the mechanism behind the unique clinical interaction between levamisole and 5-fluorouracil in colorectal carcinoma remains to be identified. Key Words: Levamisole—Duke's C colorectal carcinoma—5-Fluorouracil.
effects of levamisole on suppressed immune responses, and antitumor activity in animal tumor models (1,2). Based upon early reports demonstrating improved survival in patients with metastatic colorectal carcinoma (3), adjuvant randomized clinical trials have shown that the combination of levamisole and 5-fluorouracil (5FU) prolongs survival in patients following surgical resection of primary colorectal cancer when compared to 5FU alone (46). Levamisole by itself has not been shown to have
Levamisole is a synthetic phenylimidazothiazole that is undergoing clinical evaluation as an antineoplastic agent. Although originally used as an antihelminthic drug, oncological interest in this drug stems from early reports demonstrating restorative
Received March 26, 1991; accepted April 19, 1991. Address correspondence to Dr. J. H. Schiller at Section of Oncology, Room B5055, William S. Middleton VA Hospital, 2500 Overlook Terrace, Madison, WI 53705, U.S.A.
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a beneficial effect when compared to placebo in this patient population (7). The mechanism of action by which levamisole plus 5FU exert its antitumor activity in colorectal patients is unknown (8). Recent studies have examined the effects of levamisole and 5FU, alone or in combination, on the in vitro growth of three human colorectal cell lines. In these studies, levamisole inhibited the growth of the colon carcinoma cells only at suprapharmacologic concentrations (9). Similarly, an additive antiproliferative interaction with 5FU was evident only at suprapharmacologic doses of levamisole. These results suggest that the beneficial antitumor effects observed in clinical trials of colorectal cancer are not direct cytotoxic effects, but may be due to the immunostimulatory properties of levamisole. Early reports have suggested that levamisole may have immunomodulatory activity, particularly on increased T-cell responses to mitogens, and restoration of suppressed cellular-mediated hypersensitivity to normal (10-13). However, the effects of levamisole on monocyte and lymphocyte proliferation and cytotoxicity and other cytokine-induced cell-surface or soluble proteins are unknown. To determine the immunomodulatory properties of levamisole, we studied the in vitro effects of levamisole on monocyte and lymphocyte activation and cytotoxicity, expression of tumor-associated antigens, and induction of cytokine-induced proteins. These parameters were chosen for study because they have been postulated to play a potential role in the therapeutic activity of other biological response modifiers.
thetase), indoleamine-2,3-dioxygenase (IDO), and tumor necrosis factor (TNF). 2',5'-OHgoadenylate Synthetase (2-5A Synthetase) The activity of this enzyme was measured by incorporation of [3H]ATP into 2',5'-oligoadenylate using a method developed in our laboratory and described elsewhere (14,15). Tumor Necrosis Factor (TNF) PBMC were incubated for 24 h with or without 100 ng/ml of lipopolysaccharide (LPS) (Sigma) in the presence of increasing concentrations of levamisole. TNF in the supernatants was analyzed using a commercially available enzyme-linked immunoabsorbent assay (ELISA) (R and D Systems, Minneapolis, MN, U.S.A.). Indoleamine-2,3-Dioxygenase (IDO) The activity of IDO, which degrades tryptophan to iV-formylkynurenine, was assayed by release of [14C]formate from [I4C]tryptophan in PBMCs as previously described (16). Monocyte Assays The effects of levamisole on monocyte human leukocyte antigen (HLA) class I and II antigen expression, Fc receptor expression, and monocyte cytotoxicity were determined.
MATERIALS AND METHODS
Peripheral blood mononuclear cells (PBMCs) from healthy donors were purified from fresh heparinized blood on Ficoll-hypaque density gradients, and cultured in RPMI-1640 plus 10% fetal bovine serum (FBS) with and without levamisole at concentrations indicated in the text (Sigma Chemical Co., St. Louis, MO, U.S.A.). After 24 h, PBMCs were either harvested immediately, or frozen for later use for enzyme activity assays (2-5A synthetase and IDO). Cytokine-induced Proteins
Cytokine-induced proteins that were assessed included 2',5'-oligoadenylate synthetase (2-5A synJ Immunolher, Vol. 10, No. 5, 1991
Analysis of Cell Surface Antigens on Monocytes PBMCs that had been treated for 24 h with levamisole were incubated with specific fluorescein isothiocyanate (FITC)-labeled monoclonal antibody for HLA-DR, -DQ, and -DP (Becton Dickinson, Mountain View, CA, U.S.A.) and for Fc receptors (Medarex, Inc., New Lebanon, NH, U.S.A.) (17). Cells were analyzed by flow cytometry (FACScan, Becton Dickinson), and monocytes identified by light scatter. Nonspecific binding was compensated for by subtraction of staining by nonspecific antibody of the same isotype. Results are expressed as mean fluorescence intensity (MFI) and as percent positive cells.
Immunological Effects of Levamisole In Vitro Joan H. Schiller, *Mary Lindstrom, Patricia L. Witt, Jacquelyn A. Hank, tDavid Mahvi, Randall J. Wagner, Paul Sondel, and Ernest C. Borden Departments of Human Oncology, *Biostatistics, and tSurgery, William S. Middleton Veterans Administration Hospital and University of Wisconsin Clinical Cancer Center, Madison, Wisconsin, U.S.A.
Summary: Levamisole, an antihelminthic drug with immunological properties, has recently been reported to have antitumor activity when administered with 5-fluorouracil in patients with Duke's C colorectal carcinoma. The mechanism of this antitumor effect is unknown, but has been postulated to be related to levamisole's immunomodulatory properties. To define further the immunomodulatory activities of levamisole, we studied the in vitro effects of levamisole on monocyte and lymphocyte cytotoxicity, activation, and proliferation; induction of cytokine-induced proteins; and expression of tumor-associated antigens. Experiments utilized peripheral blood mononuclear cells from normal donors incubated in the presence of increasing concentrations of levamisole (0.1 to 100 (j-g/ml). Levamisole had no consistent effect on induction of 2',5'-oligoadenylate synthetase activity or indoleamine-2,3-dioxygenase activity, or production of tumor necrosis factor. Levamisole had no effect on monocyte cytotoxicity or expression of HLA-DR, HLA-DQ, HLA-DP, and the Fc receptor. Similarly, levamisole had no significant effect on NK or LAK cytotoxicity or the immunological activation of T-lymphocytes, assessed by expression of CD3, CD4, CD8, CD16, CD25, and CD56. Proliferation of lymphocytes from normal donors, patients with benign polyps, and patients with malignancies, with or without IL-2 or irradiated LS174T cells, was not significantly increased overall. No significant enhancement in the expression of three tumor-associated antigens (880364, NRCO-4, and ING-1) and the intercellular adhesion molecule-1 (ICAM-1) antigen on four human cancer cell lines was observed following in vitro exposure to levamisole. We conclude that levamisole is not a potent modulator of the immune parameters we examined, and that the mechanism behind the unique clinical interaction between levamisole and 5-fluorouracil in colorectal carcinoma remains to be identified. Key Words: Levamisole—Duke's C colorectal carcinoma—5-Fluorouracil.
effects of levamisole on suppressed immune responses, and antitumor activity in animal tumor models (1,2). Based upon early reports demonstrating improved survival in patients with metastatic colorectal carcinoma (3), adjuvant randomized clinical trials have shown that the combination of levamisole and 5-fluorouracil (5FU) prolongs survival in patients following surgical resection of primary colorectal cancer when compared to 5FU alone (46). Levamisole by itself has not been shown to have
Levamisole is a synthetic phenylimidazothiazole that is undergoing clinical evaluation as an antineoplastic agent. Although originally used as an antihelminthic drug, oncological interest in this drug stems from early reports demonstrating restorative
Received March 26, 1991; accepted April 19, 1991. Address correspondence to Dr. J. H. Schiller at Section of Oncology, Room B5055, William S. Middleton VA Hospital, 2500 Overlook Terrace, Madison, WI 53705, U.S.A.
297
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a beneficial effect when compared to placebo in this patient population (7). The mechanism of action by which levamisole plus 5FU exert its antitumor activity in colorectal patients is unknown (8). Recent studies have examined the effects of levamisole and 5FU, alone or in combination, on the in vitro growth of three human colorectal cell lines. In these studies, levamisole inhibited the growth of the colon carcinoma cells only at suprapharmacologic concentrations (9). Similarly, an additive antiproliferative interaction with 5FU was evident only at suprapharmacologic doses of levamisole. These results suggest that the beneficial antitumor effects observed in clinical trials of colorectal cancer are not direct cytotoxic effects, but may be due to the immunostimulatory properties of levamisole. Early reports have suggested that levamisole may have immunomodulatory activity, particularly on increased T-cell responses to mitogens, and restoration of suppressed cellular-mediated hypersensitivity to normal (10-13). However, the effects of levamisole on monocyte and lymphocyte proliferation and cytotoxicity and other cytokine-induced cell-surface or soluble proteins are unknown. To determine the immunomodulatory properties of levamisole, we studied the in vitro effects of levamisole on monocyte and lymphocyte activation and cytotoxicity, expression of tumor-associated antigens, and induction of cytokine-induced proteins. These parameters were chosen for study because they have been postulated to play a potential role in the therapeutic activity of other biological response modifiers.
thetase), indoleamine-2,3-dioxygenase (IDO), and tumor necrosis factor (TNF). 2',5'-OHgoadenylate Synthetase (2-5A Synthetase) The activity of this enzyme was measured by incorporation of [3H]ATP into 2',5'-oligoadenylate using a method developed in our laboratory and described elsewhere (14,15). Tumor Necrosis Factor (TNF) PBMC were incubated for 24 h with or without 100 ng/ml of lipopolysaccharide (LPS) (Sigma) in the presence of increasing concentrations of levamisole. TNF in the supernatants was analyzed using a commercially available enzyme-linked immunoabsorbent assay (ELISA) (R and D Systems, Minneapolis, MN, U.S.A.). Indoleamine-2,3-Dioxygenase (IDO) The activity of IDO, which degrades tryptophan to iV-formylkynurenine, was assayed by release of [14C]formate from [I4C]tryptophan in PBMCs as previously described (16). Monocyte Assays The effects of levamisole on monocyte human leukocyte antigen (HLA) class I and II antigen expression, Fc receptor expression, and monocyte cytotoxicity were determined.
MATERIALS AND METHODS
Peripheral blood mononuclear cells (PBMCs) from healthy donors were purified from fresh heparinized blood on Ficoll-hypaque density gradients, and cultured in RPMI-1640 plus 10% fetal bovine serum (FBS) with and without levamisole at concentrations indicated in the text (Sigma Chemical Co., St. Louis, MO, U.S.A.). After 24 h, PBMCs were either harvested immediately, or frozen for later use for enzyme activity assays (2-5A synthetase and IDO). Cytokine-induced Proteins
Cytokine-induced proteins that were assessed included 2',5'-oligoadenylate synthetase (2-5A synJ Immunolher, Vol. 10, No. 5, 1991
Analysis of Cell Surface Antigens on Monocytes PBMCs that had been treated for 24 h with levamisole were incubated with specific fluorescein isothiocyanate (FITC)-labeled monoclonal antibody for HLA-DR, -DQ, and -DP (Becton Dickinson, Mountain View, CA, U.S.A.) and for Fc receptors (Medarex, Inc., New Lebanon, NH, U.S.A.) (17). Cells were analyzed by flow cytometry (FACScan, Becton Dickinson), and monocytes identified by light scatter. Nonspecific binding was compensated for by subtraction of staining by nonspecific antibody of the same isotype. Results are expressed as mean fluorescence intensity (MFI) and as percent positive cells.
