IL-4 Down-Regulates Anaphylatoxin Receptors in Monocytes and Dendritic Cells and Impairs Anaphylatoxin-Induced Migration In Vivo This information is current as of September 29, 2021. Afsaneh Soruri, Ziba Kiafard, Claudia Dettmer, Joachim Riggert, Jörg Köhl and Jörg Zwirner J Immunol 2003; 170:3306-3314; ; doi: 10.4049/jimmunol.170.6.3306 http://www.jimmunol.org/content/170/6/3306 Downloaded from References This article cites 62 articles, 32 of which you can access for free at: http://www.jimmunol.org/content/170/6/3306.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology IL-4 Down-Regulates Anaphylatoxin Receptors in Monocytes and Dendritic Cells and Impairs Anaphylatoxin-Induced Migration In Vivo1 Afsaneh Soruri,* Ziba Kiafard,* Claudia Dettmer,* Joachim Riggert,† Jo¨rg Ko¨hl,‡ and Jo¨rg Zwirner2* Anaphylatoxins mobilize leukocytes to the sites of inflammation. In the present study we investigated the impact of GM-CSF, IL-4, and IFN-␥ on anaphylatoxin receptor expression in monocytes and dendritic cells (DC). IL-4 was identified as the strongest down-regulator of the receptors for C5a and C3a in monocytes and monocyte-derived DC (MoDC). To study the impact of IL-4 on anaphylatoxin-induced chemotaxis, an in vivo migration model was established. For this purpose, human monocytes and MoDC were injected i.v. into SCID mice that at the same time received anaphylatoxins into the peritoneal cavity. A peritoneal influx of Downloaded from human monocytes could be demonstrated by 4 h after injections of C5a and C3a. In line with receptor down-regulation, IL-4 treatment inhibited in vivo mobilization of human monocytes and MoDC in response to C5a and C3a. In addition to its effects on human cells, IL-4 reduced C5a receptors in murine bone marrow-derived DC and impaired recruitment of labeled bone marrow- derived DC in syngeneic BALB/c mice to i.p. injected C5a. Overall, these data suggest that inhibition of a rapid anaphylatoxin- induced mobilization of monocytes and DC to inflamed tissues represents an important anti-inflammatory activity of the Th2 cytokine IL-4. The Journal of Immunology, 2003, 170: 3306–3314. http://www.jimmunol.org/ acrophages may be activated by the Th1 cytokine The main function of DC is to collect Ags in inflamed tissues IFN-␥, resulting in up-regulation of MHC class II and and to migrate to the local lymph nodes, where specific immune M Fc␥Rs and increased production of proinflammatory responses are initiated (17). Migration of DC is critically governed cytokines such as IL-1, IL-6, TNF-␣, and chemokines (1, 2). IL-4, by the differential expression of chemokine receptors (18). Imma- a Th2 cytokine, is a counterplayer of IFN-␥ (3). Its effects on ture DC are responsive to inflammatory chemokines (19, 20), macrophages have been described as alternative activation (4), which may guide them to inflammatory sites where Ag sampling which includes induction of MHC class II and mannose receptor can take place, and maturation is induced. The maturation process, by guest on September 29, 2021 expression, but also inhibition of proinflammatory cytokine secre- which may be triggered by inflammatory stimuli such as IL-1␤, tion (e.g., IL-1, TNF-␣, IL-6, and IL-12) (5–7). With respect to TNF-␣, LPS, or CD40 ligand (CD40L) (10, 21) leads to down- down-regulation of inflammation, alternatively activated macro- regulation of receptors for inflammatory and up-regulation of re- phages are characterized by expression of anti-inflammatory cyto- ceptors for constitutive chemokines, such as macrophage inflam- kines such as IL-10 and IL-1 receptor antagonist (8, 9). matory protein 3␤ (MIP-3␤), which may induce migration of DC IL-4 is instrumental, in combination with GM-CSF, for the tran- 3 to lymphoid organs (22, 23). As a classical inflammatory stimulus, sition of monocytes into dendritic cells (DC) in vitro (10, 11). the anaphylatoxin C5a has been shown to be a chemoattractant for Monocyte-derived DC (MoDC) generated under these conditions immature DC (19). However, controversial results exist regarding are regarded as equivalent to immature DC and are widely used for the reactivity of mature DC toward C5a (24, 25). experimental as well as clinical purposes (12). IL-4 has also been Anaphylatoxins are generated by activation-induced cleavage of used for the generation of CD34ϩ cell-derived DC by some in- the third and fifth components of complement. C5a and, to a lesser vestigators (13, 14), but not by others (15, 16). extent, C3a are mediators of proinflammatory and immunoregula- tory activities (26, 27). The C3a (77aa) and C5a (74aa) peptides regulate inflammatory functions by interacting with their receptors, Departments of *Immunology and †Transfusion Medicine, Georg August University C3aR and C5aR, both of which belong to the rhodopsin family of Gottingen, Gottingen, Germany; and ‡Department of Molecular Immunology, Chil- drens Hospital Research Foundation, Cincinnati, OH 45229 seven-transmembrane, G protein-coupled receptors (28–31). Ana- phylatoxin receptors are present on myeloid and nonmyeloid leu- Received for publication September 6, 2002. Accepted for publication January 15, 2003. kocyte populations, including granulocytes and monocytes/macro- The costs of publication of this article were defrayed in part by the payment of page phages (32, 33), lymphocytes (34, 35), and DC (19, 24). Whereas charges. This article must therefore be hereby marked advertisement in accordance C5a is a potent chemotaxin for all C5aR-expressing cell types, with 18 U.S.C. Section 1734 solely to indicate this fact. C3a-induced mobilization of primary cells has only been demon- 1 This work was supported by a grant from the Deutsche Forschungsgemeinschaft (ZW 38/4-1, to J.Z.) and a stipend from Novartis, Stiftung fu¨r Klinische Forschung (to A.S.). strated for eosinophils and mast cells (36, 37). The purpose of the present study was to evaluate anaphylatoxin- 2 Address correspondence and reprint requests to Dr. Jo¨rg Zwirner, Department of Immunology, Georg August University Gottingen, Kreuzbergring 57, D-37075 Got- induced mobilization of monocytes and DC in an in vivo SCID tingen, Germany. E-mail address: [email protected] mouse model and to investigate the impact of IL-4 on anaphyla- 3 Abbreviations used in this paper: DC, dendritic cell; BmDC, bone marrow-derived toxin receptor expression and function. Our results confirmed C5a DC; C3aR, C3a receptor; C5aR, C5a receptor; C5aRA, C5a receptor antagonist; CD40L, CD40 ligand; CM, complete medium; MIP, macrophage inflammatory pro- and newly established C3a as chemoattractants for monocytes/ tein; MoDC, monocyte-derived DC. macrophages. IL-4 was found to down-regulate anaphylatoxin Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 3307 receptors in human monocytes and MoDC as well as in murine bone Flow cytometric analysis marrow-derived DC (BmDC). In parallel with receptor down-regula- For analysis of human Ags the following Abs were used: anti-HLA-DR tion, anaphylatoxin-induced mobilization was inhibited. (Ho¨lzl Diagnostics, Koln, Germany), anti-CD86, anti-CD14 (both from Dakopatts, Hamburg, Germany), and anti-CD83 (Beckman Coulter, Materials and Methods Krefeld, Germany). All Abs were either FITC- or PE-conjugated as indi- cated. Cells (2 ϫ 105) were washed with PBS containing 1.5% FCS and 10 Recombinant chemotaxins mM sodium azide. They were then blocked with heat-aggregated human ␮ ␮ Recombinant human anaphylatoxins C5a (38), C3a (39), and C3a(desArg) IgG (and murine IgG if murine cells were stained; 20 g each in 100 l) (40) were generated as described. LPS concentrations, determined by the for 20 min on ice. After washing three times with PBS containing 1.5% Limulus assay (Coatest Endotoxin; Pharmacia, Freiburg, Germany), were FCS and 10 mM sodium azide, cells were incubated with labeled Ab for 45 Ͻ20 pg LPS/␮g anaphylatoxin. Recombinant C5aR antagonist (C5aRA) min. As a negative control, FITC-conjugated IgG1/PE-conjugated IgG2a was generated as previously described (41). MIP-1␣ and MIP-3␤ were murine isotype mix was used (Dakopatts). Finally, cells were washed as obtained from PeproTech (Cell Concepts, Umkirch, Germany). described above, resuspended in PBS containing 1% formaldehyde, and analyzed in a flow cytometer (EPICS XL; Beckman Coulter, Krefeld, Ger- Monoclonal Ab against anaphylatoxin receptors many). Gating was performed to exclude dead cells from analyses. If indirect immunofluorescence staining was performed, the following The mAb hC3aRZ8 (IgG2b) was generated by immunizing BALB/c mice Abs were used: mAb anti-C5aR (hC5aRZ1), mAb anti-C3aR (hC3aRZ8), i.p. with RBL-2H3 transfectants expressing the human C3aR and fusion of irrelevant control mAb, and goat anti-mouse IgG (HϩL) FITC-conjugated spleen cells with the myeloma cell line X63-Ag8.653. Supernatants of (Dakopatts). hybridomas generated according to standard techniques were tested for their reactivity with a recombinant C3aR fragment of the large extracellular Migration of human cells in an SCID mouse model loop structure of the human C3aR and with human C3aR expressing RBL- All animal work was conducted in accordance with guidelines for the wel- Downloaded from 2H3 transfectants.