2522 zyxwvutsrqpon F. Granucci, G. Girolomoni, M. B. Lutz et al. Eur. J. Immunol. 1994. zyx 24: 2522-2526 Francesca Granucci., Giampiero GirolomoniA, Manfred B. Lutz., Maria Foti., Giulia Marconi, Paola GnocchiO, Luisa NolliO and Paola Ricciardi-Castagnoli. Department of Pharmacology., CNR Center of Cytopharmacology, University of Milan, Milan, Department of DermatologyA, University of Modena, Modena, Pharmacia-Farmitalia-Carlo Erbao, Business Area Oncology-Immunology, Nerviano and Lepetit Research Center MMDRI zyxwvutsrqp O, Gerenzano Modulation of cytokine expression in mouse dendritic cell clones* Dendritic cells (DC) play an essential role in the induction of primary immune responses; howeveqvery little information is available on cytokine production by DC. Here we determined the cytokine gene expression profile of two immortal- ized DC clones, CBI and D2SC/1, both generated from mouse spleen but differing in their activation requirements. Among the cytokines tested, only transforming growth factor-P1 was transcribed constitutively, but its production was detected only in D2SC/1 cells after treatment with granulocytelmacrophage colony-stimulating factor (GM-CSF). GM-CSF also promoted transcription and synthesis of interleukin (IL)-lB in CB1 cells that need pretreatment with GM-CSF to present major histocompatibility complex class II-restricted antigens efficiently in vitro. Lipopolysaccharide (LPS) up-regulated gene expression and induced release of tumor necrosis factor-cx in both DC clones. In addition, LPS induced transcription of IL-la and both gene expression and synthesis of IL-lP in D2SC/l cells. Interferon-y was ineffective in inducing cytokine gene expression, although it augmented the antigen-presentation capacity of DC. IL-4, IL-10 and IL-12 mRNA were not induced by any of the tested stimuli. The results suggest that DC have a limited cytokine gene expression pattern compared to macro- phages and are heterogenous in some functional properties. 1 Introduction Dendritic cells (DC) are extremely efficient in the presen- tation of MHC associated peptides and the most potent activators of naiveT lymphocytes [l]. In addition, DC play a dominant role as “professional” antigen-presenting cells (APC) for induction of Tcell priming in vivo [2-51. The features that render DC such efficient stimulators of naive Tcells are not completely known and probably include constitutive expression of high levels of MHC class zyxwvu I1 molecules and of a number of costimulatory signals such as B7.1 and B7.2, heat-stable antigen and adhesion mole- cules. Cytokines play an important role in antigen presen- tation by influencing T cell activation and directing T cell differentiation. However, very little is known about cyto- kine production by DC, most likely because DC are difficult to isolate from tissues, cannot be purified to homogeneity and have a limited life span in culture. A method for the generation of immortal functional DC lines has been recently reported [6]. These cells have the phenotypic and functional properties described for normal DC, including the ability to induceTcel1 priming in vivo [6], and, therefore, represent a good model for studying DC [I 132051 zyxwvu ~~~ ~ ~ ~~ * This work was partly supported by the Italian Association for Cancer Research (AIRC), by Biotop and by Consiglio Nazionale delle Ricerchc (ACRO project). Correspondence: Paola Ricciardi-Castagnoli, Department of Phar- macology, CNR Center of Cytopharmacology,University of Milan, Via Vanvitelli 32, 1-20129 Milan, Italy (Fax: +39-2-7490574) Abbreviations: DC: Dendritic cells LC: Langerhans cells .GM-CSF Granulocyte/macrophage-colony-stimulating factor TGF-P: Transforming growth factor p Key words: Antigen-presenting cells / Costimulatory signals / Activating signals immunobiology. In this study, we investigated cytokine mRNA expression and modulation in two distinct DC clones differing in their cytokine activation requirements for in vitro MHC class II-restricted antigen presenting function. The results showed that the cytokine gene transcription pattern was more limited compared to macro- phages and different in the two DC clones, suggesting that DC are heterogenous in some functional properties. 2 Materials and methods 2.1 Cell culture CBI dendritic cell line was derived from DBAR mice spleen primary culture and immortalized using the MIBv2Nll retroviral vector, as described [7]. D2SC/I dendritic cell line was immortalized with the same vector from BALBlc mouse spleen (manuscript in preparation). Cells were grown in Iscove’s MDM (Sigma Chemical Co., St. Louis, MO) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 pg/ml streptomycin (all from Sig- ma) and zyxwv 5% heat inactivated FCS (Boehringer Mannheim, Mannheim, Germany) at 37 “C in 5% COz, at a density of 105 cells/ml. Cells were incubated with 0.1 pg/ml lipopoly- \accharide (LPS) (Escherichia coli, B5 : 055; Sigma), I00 U/ml recombinat murine IFN-y or 200 ng/ml recombi- nant murine granulocyte/macrophage-colony-stimulating factor (GM-CSF) (Genzyme, Cambridge, MA) for I, 6 and 24 h, respectively. 2.2 RNA extraction After each incubation (I, 6,24 h), cells were collected and lysed using the guanidinium lysis solution (4 M guanidinium isothiocyanate, 25 mM sodium citrate, 0.5% sodium lauryl sarcosine, 100 mM P-mercaptoethanol). DC that were still attached to the petri dish were lysed by adding the lysis 001 4-2980/Y4/1010-2522$10.00 + .25/0 0 VCH Verlagsgesellschaft mbH, D-69451 Weinheim, 1994