Eur. J. Immunol. 1988.18: 1527-1533 Munne leishmaniasis and rGM-CSF 1527 zy Johann Greil, Barbara Bodendorfer, Martin Rollinghoff and Werner Solbach Institute for Clinical Microbiology, University of Erlangen, Erlangen Application of recombinant granulocyte-macrophage colony-stimulatingfactor has a detrimental effect in experimental murine leishmaniasis* The purpose of this study was to evaluate the effect of recombinant granulocyte- macrophage colony-stimulating factor (rGM-CSF) on BALB/c mice infected s . ~ . with the intracellular pathogen zyxwvut Leishmania major. Daily i.p. application of 1 pg rGM-CSF for 21 days following the infection led to an aggravated course of the disease in most animals. In no case was a therapeutic effect observed. In vitro analysis revealed that the parasite burden was approx. 2- to 7-fold higher in the infected lesions, in the lymph nodes draining the infection and in the spleens of rGM-CSF-treated animals than in tissues from nontreated mice. L. major-infected macrophages obtained from chronically infected mice proliferated in the presence of rGM-CSF in vitro without gaining antiparasitic effector function. However, antiparasitic effector function increased and macrophage growth was inhibited in the presence of recombinant interferon-y (IFN-y). These data indicate that rGM-CSF-induced macrophage prolif- eration alone is not sufficient to overcome infections with intracellular pathogens like L. major, since simultaneous activation of macrophages by IFN-y is required. 1 Introduction Granulocyte-macrophage colony-stimulating factor (GM- CSF) is a glycoprotein which in experimental and clinical set- tings has been shown to up-regulate the proliferation and mat- uration of granulocytes and macrophages (Ma) both in vitro and in vivo [l-31. In addition, in vitro studies provide evidence that GM-CSF can activate human and murine M a populations for antimicrobial activity against Trypanosoma cruzi [4], Sal- monella typhimurium [5], Leishmania donovani [6] and Leish- mania tropica [7]. The recent availability of sufficient amounts of recombinant GM-CSF (rGM-CSF) has evoked substantial expectations among clinicians to apply the material, first, in situations where the correction of cytopenias is required and, second, when boosting of antimicrobial host defence mediated by M@ is of importance, including infections with intracellularly grow- ing pathogens. Therefore, we studied in vivo possible antimicrobial effects of murine rGM-CSF in the model of experimental leishmaniasis of BALB/c mice. This infection model seemed to be especially attractive, since mice of this strain are exquisitely susceptible to Leishmania major, a parasite whose growth in mammalians is restricted to cells of the monocyte/M@ lineage. BALB/c mice usually succumb to the infection. This has been attrib- uted to a defect and/or dysregulation of T lymphocyte immune responses acting on M a effector cells [8-111. In particular, it [I 67641 zyxwvu * This work was supported by the Deutsche Forschungsgemeinschaft (grant Ro 325/5), it is part of the doctoral thesis of J. G. Correspondence: Werner Solbach, Institute for Clinical Microbiology, Wasserturmstr. 3, D-8520 Erlangen, FRG Abbreviations: IFN-y: Interferon-y (r)GM-CSF: (Recombinant) granulocyte-macrophage colony-stimulating factor zyxwvuts PBS: Dulbecco’s phosphate-buffered saline MTT: 3-(4,5,-Dimethylthiazol-2-y1)-2,5- diphenyl-tetrazoliumbromide Ma: Macrophage(s) LDA: Limiting dilution analysis IL 3: Interleukin 3 has been shown that the susceptibility of BALB/c mice is closely associated with a specific incapacity to generate suffi- cient amounts of interferon-y (IFN-y) in response to the infec- tion [12, 131. In vitro, IFN-y and other Ma-activating factors have been identified as important cytokines that induce M@ to effectively inhibit the growth of a variety of intracellular pathogens including L. major [14-171. The data reported here show that infected BALB/c mice treated i.p. with rGM-CSF developed a more severe disease compared to nontreated animals. No beneficial effect was observed in any of the trials. zyxw Ex vivo studies disclosed that rGM-CSF-treated mice had increased numbers of mononu- clear cells in the spleens and peritoneal cavities. Moreover the parasite burden in rGM-CSF-treated mice was significantly above that of the control animals. Parasitized splenic M a obtained from mice that were chronically infected with L. major could not be activated for an antileishamanial response by rGM-CSF in vitro. The incapacity of the cells to induce antiparasitic effector functions was not due to an intrinsic incapacity of the cells to inhibit the growth of L. major, since treatment of the M a cultures with recombinant IFN-y clearly created a potent antileishmanial effector response. 2 Materials and methods 2.1 Culture medium The culture medium was RPMI 1640 (Gibco Europe, no. 041- 02400, Karlsruhe, FRG), supplemented with L-glutamine, Hepes buffer (25 mM) and 10% fetal calf serum (FCS; Myo- clone Plus, Gibco). According to the distributors, the lipopolysaccharide (LPS) content of the culture medium was <60 pg/ml as determined in the limulus amebocyte lysate assay. 2.2 Cytokines Yeast-expressed and purified homogeneous, LPS-free (<SO pg/ml protein) rGM-CSF (lots no. 344-85-16 and 344- zy 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1988 0014-2980/88/1010-1527$02.50/0