Journal of Leukocyte Biology Volume 58, September 1995 365 Cytokine induction by the immunomodulators imiquimod and S-27609 Traci L. Testerman, John F. Gerster,* Linda M. lmbertson, Michael J. Reiter, Richard L. Miller, Sheila J. Gibson, Tamara L. Wagner, and Mark A. Tomai Department of Pharmacology and *Depanment of Ghemistiy, 3M PharmaceuticaLs, 3M Center, St. Paul, Minnesota Abstract: Imiquimod (R-.837, S-26308) and the ana- logue S-27609 were evaluated for cytokine induction in human blood cells. Both compounds induced interferon- a (IFN), tumor necrosis factor-ct (TNF), interleukin (IL)- 11, and IL-6 with S-27609 being 5 to 10 times more potent. Imiquimod and S-27609 also induced IL-la, IL-i receptor antagonist, IL- 10, granulocyte-macrophage colony.stimu- lating factor (GM-CSF), granulocyte CSF (G-CSF), and macrophage inflammatory protein-la. The profile of cy- tokines induced by imiquimod and S-27609 was different from those seen with lipopolysaccharide and polyinos- inic-polycytidylic acid. Kinetic studies with both imiqui- mod and S-27609 revealed induction of cytokines as early as 1-4 h after stimulation. Although most ofthe cytokines produced by S-27609 were secreted, significant concen- trations ofIL-lcx and IL-13 remained intracellular. Mono- cytes were largely responsible for the cytokines produced. Finally, S-27609-induced mRNA expression for TNF, IFN, and IL-8, and this induction did not require protein synthesis. Taken together, these studies extend previous findings by showing induction of additional cytokines and providing insight into the mechanism of cytokine induction by these molecules.J. Leukoc. Biol. 58: 365-372; 1995. Key Words: interferon ‘ interleukin . tumor necrosis factor lymphokines - lipopolysaccharide polynucleotides INTRODUCTION A new class of immunomodulating agents, represented by the molecule imiquimod (R-837, 5-26308), has been found to be an effective antiviral and antitumor agent in animal models. Imiquimod protected guinea pigs from infection by herpes simplex virus (HSV) when given in- travaginally, intramuscularly, intraperitoneally, intrader- mally, subcutaneously (SC), and by mouth (P0) [1, 2]. Imiquimod reduced recurrences induced in guinea pigs by latent HSV infection and, in combination with acy- clovir, significantly reduced acute HSV in guinea pigs even after lesions had developed [3-5]. Further studies indicated that imiquimod was active both prophylactically and therapeutically against cytomegalovirus infection in guinea pigs [6] and arbovirus infection in mice [7]. In addition to its antiviral properties, imiquimod inhib- ited growth of a number of murine tumors including MC-26 colon carcinoma, RIF-1 sarcoma, Lewis lung carci- noma, and a tumor induced by the chemical carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide [8, 9]. Imiqui- mod not only inhibited growth of the FCB bladder tumor in mice but actually resulted in elimination of tumors from these animals [10]. Imiquimod has no inherent antiviral or antitumor activ- ity in vitro but does stimulate secretion of interferon-a (IFN), which is partially responsible for the in vivo antivi- ral and antitumor activities. Imiquimod was first found to induce serum IFN in guinea pigs after topical, P0 or SC administration [1 1]. Further studies indicate that imiqui- mod induces IFN in other animal species including mice, rats, and monkeys [8, 12, 13]. Phase I studies in healthy adult males indicated that imiquimod given P0 was capa- ble of inducing detectable concentrations of serum IFN in these volunteers [14-16]. Using antibody neutraliza- tion, the type of IFN induced by imiquimod was identi- fled as greater than 97% IFN-a [14]. Indeed, much of the antiviral activity and antitumor activity ascribed to imiqui- mod was due to the induction of IFN, but other factors seem to contribute as well [2, 4, 8]. More recently, it was demonstrated that imiquimod, its hydroxylated metabo- lite R-842, and 5-27609 stimulated production of a number of other cytokines including tumor necrosis fac- tor-a (TNF), interleukin (IL)-lct, IL-1, IL-6, and IL-8 in cultures of human peripheral blood mononuclear cells (PBMCs) [17, 18]. Furthermore, TNF and IL-6 along with IFN were detected in the serum of mice following oral administration of imiquimod [19]. The same cytokines were produced in cultures of mouse spleen and bone marrow cells stimulated with imiquimod. In the HSV-in- fected guinea pig, imiquimod enhanced HSV-specific IL-i and IL-2 production and increased HSV-specific cell-me- diated immune responses [2]. Finally, Kono et al. [20] have demonstrated induction of IL-6 and IL-8 mRNA expression in normal and transformed human keratino- cytes. Thus, imiquimod induced a number of different cytokines and other immunomodulating effects, all of which may contribute to its overall biological activity. The cell responsible for cytokine induction in imidazo- quinoline-stimulated human PBMCs has been under in- vestigation. Megyeri et al. [21] have shown that both monocytes and B lymphocytes are capable of secreting Abbreviations: FCS, fetal calf serum; G-CSF, granulocyte colony-stimu- lating factor; GM-CSF, granulocyte-macrophage colony-stimulating fac- tor; HBSS, Hanks’ balanced salt solution; HSV, herpes simplex virus; 1FN. interferon; IL, interleukin; IL-I RA, IL-i receptor antagonist; LPS, lipopolysaccharide; MIP-i a, macrophage inflammatory protein-ia; PBMC, peripheral blood mononuclear cell; poly I:C, polyinosinic-poly- cytidylic acid; RT-PCR, reverse-transcriptase polymerase chain reaction; RT, room temperature; TNF, tumor necrosis factor-a. Reprint requests: Mark A. Tomai, 3M Pharmaceuticals, 3M Center, 270-2S-06, St. Paul, MN 55144. Received March 23, 1995; accepted May 29, 1995.