IL-4 Down-Regulates 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! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 29, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 expression in monocytes and dendritic cells (DC). IL-4 was identified as the strongest down-regulator of the receptors for C5a and in monocytes and monocyte-derived DC (MoDC). To study the impact of IL-4 on anaphylatoxin-induced , 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 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 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), 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) (5Ð7). 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 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 (28Ð31). 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), (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, ; 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 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. Immunofluorescent staining obtained with mAb fare of animals and was approved by the administration of Lower Saxony, hC3aRZ8 and mAbs anti-C3aR hC3aRZ1-4, which have been previously Germany. SCID mice (strain CB-17 scid of both sexes; 19Ð24 g) were generated by our laboratory (33), were similar (not shown). obtained from the University of Gottingen or from Charles River (Sulzfeld, The mAb hC5aRZ1 (IgG1) was generated by immunizing BALB/c mice Germany). Human cells (1 ϫ 107) were resuspended in 200 ␮l of PBS and with RBL-2H3 transfectants expressing the human C5aR. Supernatants of injected into the tail vein of SCID mice. In parallel, 200 ␮l of PBS con- hybridomas were tested for their reactivity with peptides representing the taining recombinant anaphylatoxin (2.5Ð40 ␮g) was injected i.p. After 2, 4, N-terminal 31 aa of the C5aR and with C5aR expressing RBL-2H3 trans-

24, or 48 h, mice were sacrificed, and cells were harvested from the peri- http://www.jimmunol.org/ fectants. In additional experiments mAb C5aRZ1 was shown to block bind- toneum by flushing with 7 ml of PBS and counted using a hemocytometer. ing of fluorescein-conjugated recombinant C5a to its receptor on RBL-2H3 Thereafter, migrated human monocytes, macrophages, or MoDC were transfectants. stained with PE-conjugated anti-HLA-DR (alone and in combination with FITC-anti-CD86, FITC-anti-CD83, or FITC-anti-CD14) and analyzed by Human monocytes/macrophages and MoDC flow cytometry. Absolute numbers of migrated human cells were calcu- lated from the percentage of HLA-DRϩ cells and the total peritoneal cell Leukocytes were obtained by leukapheresis from volunteer blood donors at ϩ ϩ University Clinic Gottingen. PBMC were isolated by centrifugation on a count. Human or murine B (CD19 ) and T (CD3 ) lymphocytes were Ficoll-Hypaque discontinuous gradient. PBMC were cultured for1hat1ϫ never detectable in the peritoneal cavity of SCID mice. 7 107 cells/ml in endotoxin-free RPMI 1640 (Biochrom, Berlin, Germany) To block migration, human cells (1 ϫ 10 ) were incubated with 50 ␮g supplemented with 5% heat-inactivated autologous serum in flat-bottom of mAb (anti-C5aR hC5aRZ1, anti-C3aR hC3aRZ8) in 1 ml of RPMI for plates. After washing off nonadherent cells, adherent PBMC (Ͼ80% mono- 1 h on ice. Cells were washed once, transferred into PBS, and injected i.v. by guest on September 29, 2021 cytes) were cultured in RPMI 1640 supplemented with 10% FCS (PAN Migration of murine BmDC in vivo Biotech, Aidenbach, Germany), penicillin/streptomycin, L-glutamine, GM- CSF (300 U/ml), and IL-4 (300 U/ml; both from R&D Systems, Wiesba- Murine BmDC were labeled with the red fluorescent dye PKH-26 (Sigma- den, Germany). After 5Ð7 days, cultured MoDC expressed HLA-DR, but Aldrich, Deisenhofen, Germany) according to the manufacturer’s instruc- not CD14, and were characterized as immature due to their moderate ex- tions. Labeled BmDC (1 ϫ 107 in 200 ␮l of PBS) were injected into the pression of CD86 and low expression of CD83. Further differentiation into tail vein of BALB/c mice (weight, 25Ð29 g; age, 8Ð20 wk). Immediately mature DC was accomplished by incubation with CD40L/enhancer (100 ng/ thereafter, the chemotaxin (10 ␮gin200␮l; in some experiments 5Ð40 ␮g ml; Alexis, San Diego, CA) plus TNF-␣ (25 ng/ml; R&D Systems) for 48 h. as indicated) was injected into the peritoneal cavity. Two to 48 h later, mice To investigate the effects of saturating concentrations of GM-CSF (300 were killed, and peritoneal lavage was performed using 10 ml of PBS. U/ml), IL-4 (300 U/ml), and IFN-␥ (300 U/ml; all from R&D Systems) on Subsequently, peritoneal cells were counted using a hemocytometer and anaphylatoxin receptor expression in human monocytes, adherent PBMC analyzed by FACS. Absolute numbers of migrated BmDC were calculated were cultured in RPMI with 10% FCS for 2 days. To investigate the effect from the percentage of red fluorescent cells and the total peritoneal cell of IL-4 on MoDC, adherent PBMC were cultured in RPMI 1640 with 10% count. Red-labeled granulocytes or lymphocytes that might contaminate FCS in the presence of saturating concentrations of GM-CSF (300 U/ml) BmDC preparations to some degree were never observed in the peritoneal with or without IL-4 (300 U/ml) for 5 days. To obtain macrophages, ad- cavity of injected BALB/c mice. herent PBMC were cultured in the presence of 5% pooled human serum for 2 or 4 days. RT-PCR Murine BmDC Total RNA was isolated using a commercially available preparation method (NucleoSpin; Macherey-Nagel, Duren, Germany). RNA was di- A modified method of Inaba et al. (15) for the isolation of bone marrow gested with DNase I to exclude genomic DNA contamination. First-strand precursors was used. Briefly, bone marrow was collected from tibias and cDNA synthesis on 2Ð5 ␮g of RNA was performed using the SuperScript femurs of female BALB/c mice, passed through a nylon mesh to remove Preamplification Kit (Life Technologies, Eggenstein, Germany) with ran- small pieces of bone and debris, resuspended in complete medium (CM: dom hexanucleotide primers. The PCR mixture (50 ␮l) contained varying RPMI 1640 containing 5% FCS, 20 ␮g/ml gentamicin, 0.1 mM nonessen- amounts of cDNA, 0.2 mM dNTP, 0.5 ␮M5Ј and 3Ј oligonucleotide prim- tial amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 ␮M ers as well as reaction buffer and 1.25 U of DNA polymerase (HotStarTaq; 2-ME), and cultured in tissue culture dishes for 2 h. Nonadherent cells were Qiagen, Hilden, Germany). DNA amplification was conducted using a ther- collected, and aliquots of 1 ϫ 106 cells were placed in 24-well plates mocycler (Mastercycler; Eppendorf, Hamburg, Germany). PCR conditions containing 1 ml of CM with two different combination of cytokines: 100 were 28 cycles of 94¡C (1 min), 59¡C (1 min), and 72¡C (1 min) for human U/ml GM-CSF with or without 200 U/ml IL-4 (both from Cell Concepts). ␤-actin; 32 cycles of 94¡C (1 min), 59¡C (1 min), and 72¡C (1 min) for Two-thirds of the medium was replaced on days 3 and 5. On day 6 of human C5a and C3a receptors; 28 cycles of 94¡C (1 min), 50¡C (1 min), culture, nonadherent cells were transferred into six-well plates in CM (1 ϫ and 72¡C (1 min) for murine ␤-actin; and 27 cycles of 94¡C (1 min), 67¡C 106 cells/ml) with cytokines and maintained for 2 additional days. Both (1 min), and 72¡C (1 min) for murine C5aR. The following oligonucleotide BmDC preparations were immature, as characterized by their low expres- primer sets were obtained from MWG-Biotech (Edersberg, Germany): sion of MHC class II I-E, CD80, and CD86 and low stimulation of allo- human ␤-actin: sense, 5Ј-AAGGCCAACCGCGAGAAGATGA; and anti- geneic T lymphocytes, but could be differentiated with LPS into mature, sense, 5Ј-GGAAGAGTGCCTCAGGGCAGCG (amplifying a 451-bp frag- highly stimulatory DC (data not shown). ment) (42); human C5aR: sense, 5Ј-CAGGAGACCAGAACATGA 3308 IL-4 DOWN-REGULATES ANAPHYLATOXIN RECEPTORS

receptor expression over time. Next, we analyzed the impact of IL-4 on monocytes cultured with GM-CSF for 5 days. As shown in Fig. 2, IL-4 distinctly down-regulated C5aR and C3aR numbers and mRNA compared with monocytes cultured in the presence of GM-CSF alone. IL-4- plus GM-CSF-treated monocytes are considered immature DC.

In vivo migration of human monocytes and DC in an SCID mouse model To investigate anaphylatoxin-induced migration in vivo, an SCID mouse model was established. After i.v. injection of freshly iso- lated human monocytes or MoDC and i.p. injection of C5a (10 ␮g), mice were killed 2, 4, 24, or 48 h later, and peritoneal cells were analyzed. Table II demonstrates that human monocytes were abundantly present in the peritoneal cavity as early as 4 h after their injection, whereas MoDC could be detected i.p. only after 24 h. We then explored the impact of increasing amounts of C5a on the peritoneal influx of monocytes or macrophages 4 and 24 h after

FIGURE 1. Regulation of C5aR and C3aR expression in monocytes. their injection (Fig. 3). Migration of these cells was inducible by 5 Downloaded from Monocytes were cultured for 2 days in FCS (A and C) or human serum (B) and 10 ␮g of C5a at both time points investigated, whereas 2.5 ␮g in the presence or the absence (control) of cytokines. Cells were analyzed was ineffective. C3a also recruited human monocytes/macro- for the expression of C5aR and C3aR by indirect immunofluorescence phages into the peritoneal cavity; however, distinctly higher quan- staining and FACS. The mean Ϯ SE of at least five independent experi- tities, compared with those of C5a, were required. The C-termi- ments are shown. D, RT-PCR analysis of C5aR and ␤-actin mRNA in monocytes cultured for 2 days in the presence and the absence of IL-4 is nally desarginated catabolite of C3a, C3a(desArg), was completely demonstrated. ineffective as a chemoattractant for monocytes/macrophages using http://www.jimmunol.org/ up to 40 ␮g/animal. Furthermore, monocytes/macrophages did not migrate i.p. if PBS was injected (n ϭ 6; data not shown). ACTCC; and antisense, 5Ј-TACATGTTGAGCAGGATGAGGG (ampli- We also investigated whether C5a-mediated recruitment in vivo fying a 376-bp fragment) (29); human C3aR: sense, 5Ј-CAGACAG was dependent on signaling through the C5aR. When human GACTCGTGGAGAC; and antisense, 5Ј-GACAATGATGGAGGGGAT GAG (amplifying a 381-bp fragment) (43); murine ␤-actin: sense, 5Ј- monocytes were preincubated in vitro with anti-C5aR mAb spe- TGGAATCCTGTGGCATCCATGAAAC; and antisense, 5Ј-TAAAACGC cifically blocking the binding of C5a to human C5aR, their mobi- AGCTCAGTAACAGTCCG (amplifying a 348-bp fragment) (44); murine lization was abrogated (Table III). However, binding of anti-C5aR C5aR: sense, 5Ј-AAGGTCCGCGGGACTGGCCTGG; and antisense, 5Ј- mAb did not impede the ability of monocytes to migrate in re- GAGAGCGTTTCGTATGATGCTGGGG (amplifying a 536-bp fragment) sponse to MIP-1␣. Furthermore, preincubation with anti-C3aR by guest on September 29, 2021 (45). PCR products (10 ␮l) were separated by electrophoresis in 1.2Ð1.5% agarose gels and visualized by UV light illumination after ethidium bro- mAb was without influence on C5a-induced peritoneal influx of mide staining. To control for saturation effects of the PCR reaction, pre- monocytes. Thus, the possibility was excluded that anti-C5aR liminary experiments were assayed at different cycle numbers by removing mAb blocks cell migration unspecifically. These control experi- part of the reaction at appropriate times. ments also demonstrate that the C5a does not exert its chemotactic effects by inducing the release of secondary chemotactic sub- Results stances from resident peritoneal cells. Anaphylatoxin receptor expression in human monocytes and DC Reagents to specifically block C3aR-mediated activities are not GM-CSF and IL-4 are commonly used for the generation of DC available. To inhibit chemotaxis of monocytes in a C3aR-specific from monocytes. We therefore investigated the impact of GM-CSF manner, we took advantage of the reported internalization of ana- and IL-4 compared with IFN-␥ on C5aR and C3aR expression in phylatoxin receptors upon ligand exposure. Preincubation of monocytes after a 2-day incubation period. The strongest down- monocytes overnight with C3a completely blocked their C3a-in- regulation of C5aR and C3aR proteins as detected by mAbs was duced migration 4 and 24 h after injection, whereas C5a-induced seen for IL-4 and IL-4 plus GM-CSF, whereas GM-CSF and migration was unaffected (Fig. 4A). Vice versa, if monocytes were IFN-␥ alone were less potent (Fig. 1). IL-4 also down-regulated preincubated with C5a, C5a-induced, but not C3a-induced, migra- C5aR mRNA, as documented by RT-PCR (Fig. 1). The negative tion was inhibited. Preincubation of monocytes with pertussis impact of IL-4 on C5aR number was independent of the source of toxin also abolished migration toward anaphylatoxins in vivo, serum used, as it was also observed in the presence of human which confirmed the involvement of G proteins in signal transduc- serum (Fig. 1). Table I shows the impact of IL-4 on anaphylatoxin tion (Fig. 4B).

Table I. C5aR and C3aR expression in monocytes over timea

C5aR C3aR

Day 1 Day 2 Day 3 Day 6 Day 1 Day 2 Day 3 Day 6

GM-CSF 144 147 102 74 11 16 15 12 IL-4 121 69 33 26 9.6 3.8 2.4 1.8 GM-CSF ϩ IL-4 106 60 29 11 7.2 3.3 1.1 1.2

a Human monocytes were cultured for 1, 2, 3, and 6 days in medium containing FCS in the presence of GM-CSF or IL-4. Cells were then analyzed for the expression of anaphylatoxin receptors by indirect immunofluorescence staining and FACS. The mean fluorescence intensity values of one experiment of two performed are shown. The Journal of Immunology 3309

to 20 ␮g of C5a was ligand-specific, as it could be abolished by C5aR blockade. IL-4 also delayed the migration of MoDC in response to ana- phylatoxins (Fig. 6). Following peritoneal C5a (10 ␮g) injection, MoDC were undetectable after 4 h, whereas after 24 h, migration was evident (Fig. 6A). Increasing the amount of C5a to 20 ␮g partially restored the responsiveness of MoDC, as cells were de- tectable i.p. even 4 h after their injection. In contrast to C5a, C3a up to 40 ␮g was not effective in mobilizing MoDC into the peri- toneal cavity of SCID mice. Fig. 6B demonstrates that it was IL-4 that impaired anaphylatoxin-induced mobilization of MoDC in vivo. Whereas 5 ␮g of C5a mobilized GM-CSF-treated monocytes into the peritoneal cavity of SCID mice 24 h after their injection, this amount of C5a was ineffective in attracting cells that had been cultured with IL-4 (representing MoDC). IL-4 treatment also abol- ished the reactivity of MoDC to C3a (Fig. 6B). Mobilization of injected MoDC was ligand specific, as it was inhibitable by C5aR blockade (Table III). Furthermore, no migra-

tion of MoDC was seen if PBS was injected (n ϭ 3; data not Downloaded from shown).

Impact of maturation on the in vivo migration of DC Incubation of MoDC in the presence of CD40L and TNF-␣ re- sulted in phenotypic and functional maturation, as evidenced by

the strong up-regulation of CD83, CD86, and HLA-DR and the http://www.jimmunol.org/ ability to stimulate allogeneic lymphocytes (data not shown). The maturation process impaired the ability of MoDC to migrate in vivo in response to C5a, as shown by the absence of MoDC in the peritoneal cavity 24 h after C5a (10 ␮g) injection (Fig. 7). Instead, mature MoDC gained responsiveness to MIP-3␤. Migration of monocytes (n ϭ 4) and immature MoDC (n ϭ 4) in response to MIP-3␤ (10 ␮g) was not observed (data not shown).

In vivo migration of BmDC in syngeneic BALB/c mice by guest on September 29, 2021 Labeled murine GM-CSF-treated BmDC that were injected into the tail vein of BALB/c mice migrated into the peritoneal cavity in response to local injections of the anaphylatoxin C5a. Peritoneal accumulation of BmDC could be measured as early as 2 h after the injection of C5a and was still substantial after 48 h (Fig. 8A). Migration of BmDC to C5a was dependent on its interaction with the C5aR. Preincubation of BmDC with C5aRA, which spe- cifically blocks binding of the ligand to its receptor, abolished in vivo migration in response to C5a (Fig. 8B).

Impact of IL-4 on C5a-induced recruitment of BmDC in vivo FIGURE 2. IL-4 down-regulates anaphylatoxin receptor expression in Murine BmDC lost their ability to migrate in response to C5a (10 MoDC. Monocytes were cultured for 5 days in GM-CSF with or without ␮g) in vivo when they were cultured with IL-4. Neither 4 nor 24 h IL-4 and were analyzed by FACS. A, Dot plots show double-immunola- after the injection of C5a were IL-4-treated BmDC detectable in beled cells. B, Histograms show cells stained with mAbs against C3aR and the peritoneal cavity, with the exception of one experiment in C5aR by indirect immunofluorescence. Data are representative of three independent experiments. C, RT-PCR analysis of ␤-actin, C3aR, and C5aR in monocytes cultured for 5 days in GM-CSF with or without IL-4 is shown. Table II. Peritoneal mobilization of human monocytes and MoDC to C5aa

Monocytesb MoDC

Impact of IL-4 on anaphylatoxin-induced in vivo migration of 2h 0 0 monocytes and DC 4 h 339,000 314,600 0 0 24 h 372,900 748,000 228,600 289,700 We investigated the functional consequences of IL-4-induced 48 h 7,200 110,200 69,500 9,300 C5aR and C3aR down-regulation in monocytes in the SCID mouse a Human monocytes and MoDC (1 ϫ 107) were injected i.v., and C5a (10 ␮g) was model. IL-4 treatment abolished cell migration in response to C5a injected i.p. into SCID mice. Two, 4, 24, or 48 h later, peritoneal cells were recovered (10 ␮g) or C3a (40 ␮g) as measured 4 h after their injection (Fig. by lavage, counted, and stained with PE-conjugated anti-HLA-DR. The absolute num- bers of migrated HLA-DRϩ cells were calculated from the percentage of red fluo- 5A). Unresponsiveness of IL-4-treated monocytes could be over- rescent cells as determined by FACS and the total peritoneal cell count. Two inde- come by injecting 20 ␮g of C5a (Fig. 5B). Migration in response pendent experiments for each cell type are shown. 3310 IL-4 DOWN-REGULATES ANAPHYLATOXIN RECEPTORS

FIGURE 3. Dose-dependent migra- tion of monocytes and macrophages in vivo. Freshly isolated monocytes and macrophages cultured for 2 and 4 days, respectively, were injected i.v. into SCID mice that at the same time re- ceived C5a (2.5, 5, or 10 ␮g) or C3a (10, 20, or 40 ␮g) i.p. 4 or 24 h later, peritoneal cells were harvested, and hu- man cells were analyzed by FACS after Downloaded from staining with PE-conjugated anti-HLA-DR and FITC-conjugated anti-CD14. A, Abso- lute numbers of migrated HLA-DRϩ hu- man cells were calculated from the percent- age of red fluorescent cells and the total peritoneal cell count; B, dot plot analyses of http://www.jimmunol.org/ double-labeled human cells are shown. by guest on September 29, 2021

which 9200 labeled cells were counted (Fig. 9A). Parallel to its that were cultured in the presence of GM-CSF alone was observed negative impact on C5aR-mediated chemotaxis, IL-4 treatment with as little as 5 ␮g of C5a after 4 h. also down-modulated the expression of C5aR mRNA in BmDC Peritoneal influx of BmDC (treated with GM-CSF with or with- (Fig. 9B). out IL-4) was never observed in the absence of anaphylatoxin chal- We also examined whether higher doses of C5a could overcome lenge (n ϭ 16), excluding the possibility that BmDC spontane- the unresponsiveness of IL-4-treated BmDC toward the anaphyla- ously migrated i.p. toxin C5a in vivo. Fig. 10 demonstrates that this was indeed the case. When IL-4-treated BmDC were harvested 4 h after injection, Discussion 40 ␮g of C5a were required to induce a peritoneal influx of labeled One of the main functions of anaphylatoxins is the recruitment of BmDC. After 24 h, 20 ␮g of C5a were required to induced the leukocytes to the sites of infection, inflammation, and trauma. Che- migration of BmDC. In contrast, peritoneal mobilization of BmDC motactic responses of monocytes and macrophages to C5a have

Table III. Blockade of the C5a-induced peritoneal mobilization of monocytes and MoDCa

Time (h) C5a C5a ϩ hC5aRZ1b C5a ϩ hC3aRZ8b MIP-1␣ ϩ hC5aRZ1b

Monocytes 4 157,400c 0 200,200 Monocytes 4 144,750 0 Monocytes 24 630,300 0 681,300 MoDC 24 97,200 0 91,300 MoDC 24 1,032,200 0 745,200 MoDC 24 176,700 0 178,360

a Human monocytes and MoDC (1 ϫ 107) were injected i.v., and C5a or MIP-1␣ (10 ␮g) was injected i.p. into SCID mice. Four or 24 h later, peritoneal cells were collected, counted, and stained with PE-conjugated anti-HLA-DR. b Human cells were preincubated with anti-C5aR or anti-C3aR mAbs before injection. c Absolute numbers of migrated HLA-DRϩ cells were calculated from the percentage of red fluorescent cells as determined by FACS and the total peritoneal cell count. The Journal of Immunology 3311

FIGURE 4. Preincubation with anaphylatoxins and pertussis toxin blocks C5a- and C3a-mediated migration of macrophages in vivo. Macro- phages (cultured for 4 days in 5% human serum) were incubated overnight FIGURE 6. IL-4 inhibits migration of MoDC. A, MoDC cultured in Downloaded from in the presence or the absence of anaphylatoxin (2 ␮g/ml; A) or pertussis GM-CSF and IL-4 were injected i.v. into SCID mice. After 4 or 24 h, toxin (100 ng/ml; B). Cells were then injected into SCID mice i.v. that at peritoneal cells were harvested, counted, and stained with PE-anti-HLA- the same time received C3a or C5a i.p. After 24 h, peritoneal cells were DR. The mean Ϯ SE of at least four independent experiments are shown. harvested, counted, and stained with PE-anti-HLA-DR. Absolute numbers B, After culturing monocytes for 5 days with GM-CSF or GM-CSF plus of migrated HLA-DRϩ cells were calculated from the percentage of red IL-4 (MoDC), cells were injected into SCID mice i.v., which at the same fluorescent cells determined by FACS analysis and the total peritoneal cell time received C5a or C3a i.p. After 24 h, peritoneal cells were harvested, counted, and stained with PE-anti-HLA-DR. The mean values of two in- count. The mean values of two independent experiments are shown. http://www.jimmunol.org/ dependent experiments are shown. Absolute numbers of migrated HLA- DRϩ cells were calculated from the percentage of red fluorescent cells as been described in the past (46, 47). However, no such report exists determined by FACS and the total peritoneal cell count. for C3a, and only the murine macrophage cell line J774A.1 could be mobilized by C3a gradients in vitro (48). This may not be sur- prising, as primary monocytes express ϳ6 times more C5aR than Human monocytes injected i.v. accumulated in the peritoneal cav- C3aR molecules on their surface (33). Our laboratory has been ity if C3a was present. Compared with C5a, higher amounts of C3a unable to detect directed migration of monocytes/macrophages to had to be injected to attract similar cell numbers. This observation can be explained by differences in absolute anaphylatoxin receptor C3a using a standard in vitro microchemotaxis assay and polycar- by guest on September 29, 2021 bonate membranes (J. Zwirner, unpublished observations). This in numbers on monocytes (33) and by carboxypeptidase-mediated vitro approach has been shown to be hampered by a lack of sen- removal of the terminal arginine in vivo, which completely inac- sitivity and may not permit discrimination between chemotactic tivates C3a, but not C5a (51). It was therefore not surprising that and chemokinetic cell motilities, since cell velocities cannot be the catabolite of C3a, C3a(desArg), was ineffective in attracting calculated (49, 50). monocytes/macrophages in vivo. The ubiquitous carboxypeptidase Therefore, in the present study we investigated migration in vivo N may constitute an essential regulatory mechanism to control C3a using immunodeficient SCID mice as recipients of human cells. activity in vivo, as there is 10 times more C3 than C5 present in serum (51). Of note, a spontaneous random influx of human mono- cytes/macrophages into the peritoneal cavity in the absence of ana- phylatoxins did not occur. Therefore, anaphylatoxin-induced mo- bilization in the SCID mouse model appears to be exclusively chemotactic in nature. Agonist-induced, rapid receptor desensitization and internaliza- tion are important control mechanisms described for G protein- coupled receptors, including C3aR and C5aR (52, 53). If agonist

FIGURE 5. IL-4 inhibits anaphylatoxin-mediated migration of mono- cytes. A, Monocytes were cultured for 2 days in the presence or the ab- sence of IL-4. Cells were then injected into SCID mice i.v. that at the same FIGURE 7. Mature MoDC gain responsiveness to MIP-3␤. Mature time received C3a or C5a i.p. After 4 h, peritoneal cells were harvested, MoDC were generated by culturing monocytes for 5 days with GM-CSF counted, and stained with PE-anti-HLA-DR. Absolute numbers of mi- and IL-4 and for 2 additional days with TNF-␣ and CD40L. Cells were grated HLA-DRϩ cells were calculated from the percentage of red fluo- injected i.v. into SCID mice that at the same time received C5a (10 ␮g) or rescent cells as analyzed by FACS and the total peritoneal cell count. The MIP-3␤ (10 ␮g) i.p. 24 h later, peritoneal cells were harvested and human mean Ϯ SE of three independent experiments are shown. B, Migration of cells were identified by PE-anti-HLA-DR in combination with FITC-anti- IL-4-treated monocytes in response to 20 ␮g of C5a in vivo could be CD86 or FITC-anti-CD83. One representative of four independent exper- blocked by preincubation with mAb anti-C5aR hC5aRZ1. iments is shown. 3312 IL-4 DOWN-REGULATES ANAPHYLATOXIN RECEPTORS

FIGURE 8. Migration of GM-CSF-treated murine BmDC to C5a in vivo is time dependent and specific. BmDC were labeled with the red fluorescent dye PKH-26 and injected i.v. (1 ϫ 107) together with 10 ␮g of C5a i.p. into BALB/c mice. A, Peritoneal cells were harvested by lavage 1, 2, 3, or 4 h, and 4, 24, or 48 h later, counted, and analyzed by FACS. Absolute num- bers of migrated BmDC were calculated from the per- centage of red fluorescent cells and the total peritoneal cell count. B, FACS histograms show labeled BmDC before injection and peritoneal exudate cells 4 h after BmDC (untreated or treated with C5aRA) injection i.v. together with C5a i.p. Downloaded from exposure is prolonged, longer-lasting down-regulation has been C5a is a potent proinflammatory stimulus that attracts and de- shown for some G protein-coupled receptors (54). We now dem- granulates monocytes/macrophages as well as induces them to pro- onstrate that prolonged exposure of monocytes to C3a inhibited duce IL-1␤, IL-6, IL-8, and TNF-␣ (59Ð61). The proinflammatory migration in vivo in response to C3a for at least 24 h, whereas activity of monocytes following exposure to C3a is less evident. preincubation with C5a did not affect C3a-induced mobilization Human monocytes responded to C3a with an increase in intracel- http://www.jimmunol.org/ and vice versa. These data suggest a ligand-specific functional lular calcium ions (39). Furthermore, the murine macrophage cell down-regulation of anaphylatoxin receptors. It may serve to limit line J774A.1 migrated to C3a gradients in vitro (48). Our results excessive proinflammatory activities of anaphylatoxins during now demonstrate for the first time that a complex cellular response continuous complement activation in vivo. such as migration in vivo can be induced in monocytes via C3aR- IL-4-treatment down-regulated anaphylatoxin receptors in mediated activation. Inhibition of C3a-induced, in addition to C5a- monocytes and inhibited their mobilization in response to anaphy- induced mobilization of monocytes in vivo may thus represent an latoxins in vivo. In contrast, IL-4 has been shown recently to up- important anti-inflammatory activity of IL-4. regulate IL-8Rs (CXCR1 and CXCR2) in human monocytes, IL-4 plays a central role in the generation of DC in vitro, in which suggests that IL-8 contributes to the accumulation and po- particular if monocytes are used as progenitor cells (10, 11). In the by guest on September 29, 2021 sitioning of mononuclear in Th2-dominated responses present study we observed that IL-4 is a negative regulator of (55). Vice versa, the regulatory effect of IL-4 on C5aR expression numbers and function of anaphylatoxin receptors in MoDC. How- may be linked to the down-regulation of Th1 responses. Indeed, ever, C5a-induced migration of MoDC has been documented by blockade of C5aR rendered human monocytes unable to produce other groups using in vitro chemotaxis assays (19, 25). Our data IL-12 (56). It is tempting to speculate that during Th2 responses, IL-4 confirm these results, as MoDC also migrated in response to C5a prevents the recruitment of monocytes and the production of IL-12 in vivo; however, IL-4 treatment considerably reduced the sensi- through C5aR down-regulation. On the other hand, C5a has been tivity of DC to C5a stimulation and abrogated their responsiveness shown to suppress IL-12 production in stimulated monocytes (57, 58). to C3a. IL-4 is also included in protocols for the generation of DC Additional experiments are needed to reconcile these conflicting from bone marrow progenitor cells, although its use may not be results. obligatory. As shown for MoDC, IL-4 exerted a negative regula- tory impact on C5aR expression in BmDC and distinctly impaired their ability to migrate to C5a. One may speculate that monocyte- or bone marrow-derived DC generated in the presence of IL-4 in

FIGURE 9. IL-4 inhibits migration of BmDC and down-regulates C5aR mRNA. A, Murine BmDC treated with GM-CSF in the presence or the absence of IL-4 were labeled with the red fluorescent dye PKH-26 and FIGURE 10. Increasing amounts of C5a overcome IL-4-induced unre- injected i.v. (1 ϫ 107) together with 10 ␮g of C5a i.p. into BALB/c mice. sponsiveness of murine BmDC to C5a in vivo. GM-CSF-treated and GM- Peritoneal cells were harvested 4 or 24 h later, counted, and analyzed by CSF- plus IL-4 treated BmDC were labeled with the red fluorescent dye FACS. Absolute numbers of migrated BmDC were calculated from the PKH-26 and injected i.v. (1 ϫ 107 cells) together with varying amounts of percentage of migrated labeled cells and the total peritoneal cell count. The C5a i.p. into BALB/c mice. Peritoneal cells were collected 4 or 24 h after mean Ϯ SE of at least six independent experiments are shown. B, RT-PCR C5a injection, counted, and analyzed by FACS. Absolute numbers of mi- results for ␤-actin and C5aR in BmDC cultured in the absence or the grated BmDC were calculated from the percentage of red fluorescent cells presence of IL-4 are shown. and the total peritoneal cell count. The Journal of Immunology 3313 vitro do not represent the equivalent of inflammatory DC in vivo, 16. Lutz, M. B., N. Kukutsch, A. L. J. Ogilvie, S. Ra¬ssner, F. Koch, N. Romani, and as rapid influx of DC is a hallmark of the acute inflammatory G. Schuler. 1999. An advanced method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J. Immunol. Methods 223:77. response in vivo (62). This hypothesis is supported by our recent 17. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of finding that M-DC8ϩ, blood-derived, immature DC represent an . Nature 392:245. 18. Caux, C., L. Ait-Yahia, K. Chemoin, O. De Bouteiller, M. C. Dieu-Nosjean, inflammatory type of DC that responds to C5a with a prompt and B. Homey, C. Massacrier, B. Vanbervliet, A. Zlotnik, and A. Vicari. 2000. Den- robust mobilization in the SCID mouse model (63). dritic cell biology and regulation of dendritic cell trafficking by chemokines. C5a has been suggested to be involved in the trafficking of im- Springer Semin. Immunpathol. 22:345. 19. Sozzani, S., F. Sallusto, W. Luini, D. Zhou, L. Piemonti, P. Allavena, mature as well as mature DC, as both cell types expressed equal J. V. Damme, S. Valitutti, A. Lanzavecchia, and A. Mantovani. 1995. Migration numbers of C5aR and showed no differences in their ability to of dendritic cells in response to formyl peptides, C5a, and a distinct set of che- migrate to C5a in vitro (25). We now demonstrate that mature mokines. J. Immunol. 155:3292. 20. Sozzani, S., W. Luini, A. Borsatti, N. Polentarutti, D. Zhou, L. Piemonti, G. MoDC are further inhibited in their ability to migrate to C5a com- D’Amico, C. A. Power, T. N. Wells, M. Gobbi, et al. 1997. Receptor expression pared with immature DC, whereas they gained responsiveness to and responsiveness of human dendritic cells to a defined set of CC and CXC MIP-3␤. A possible explanation for the discrepant results may be chemokines. J. Immunol. 159:1993. 21. Sallusto, F., and A. Lanzavecchia. 1995. Dendritic cells use macropinocytosis that IL-4-treated DC already express drastically reduced C5aR and the mannose receptor to concentrate in the MHC class II compart- numbers compared with DC cultured in the absence of IL-4. A ment: downregulation by cytokines and bacterial products. J. Exp. Med. 182:389. 22. Gunn, M. D, K. Tangemann, C. Tam, J. G. Cyster, S. D. Rosen, and further reduction of receptor numbers by DC maturation may be L. T. Williams. 1998. A chemokine expressed in lymphoid high endothelial susceptible to minor variations in culture conditions and thus not venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proc. always be measurable. Natl. Acad. Sci. USA 95:258. 23. Kellermann, S. A, S. Hudak, E. R. Oldham, Y. J. Liu, and L. M. McEvoy. 1999. In summary, this study shows that C3a recruits monocytes/mac- The CC chemokine receptor-7 ligands 6Ckine and macrophage inflammatory Downloaded from rophages in a receptor-specific manner in vivo, although it is not as protein-3␤ are potent chemoattractants for in vitro- and in vivo-derived dendritic effective as C5a. Impairment of anaphylatoxin-induced mobiliza- cells. J. Immunol. 162:3859. 24. Kirchhoff, K., O. Weinmann, J. Zwirner, G. Begemann, O. Go¬tze, A. Kapp, and tion of monocytes and DC by IL-4 correlates with down-regulation T. Werfel. 2001. Detection of anaphylatoxin receptors on CD83ϩ dendritic cells of anaphylatoxin receptors. We suggest that inhibition of a rapid derived from human skin. Immunology 103:210. recruitment of monocytes and DC represents an important anti- 25. Yang, D., Q. Chen, S. Stoll, X. Chen, O. M. Z. Howard, and J. J. Oppenheim. 2000. Differential regulation of responsiveness to fMLP and C5a upon dendritic inflammatory activity of the Th2 cytokine IL-4. cell maturation: correlation with receptor expression. J. Immunol. 165:2694. http://www.jimmunol.org/ 26. Gerard, C., and N. P. Gerard. 1994. C5a anaphylatoxin and its seven transmem- brane-segment receptor. Annu. Rev. Immunol. 12:775. Acknowledgments 27. Cooper, N. R. 1999. Biology of the . In Inflammation: Basic We thank Jutta Wollenweber and Ines Heine for their excellent technical principles and clinical correlates. J. I. Gallin, and R. Snyderman, eds. Lippincott assistance. Williams & Wilkins, Philadelphia, p. 281. 28. Gerard, N. P., and C. Gerard. 1991. The chemotactic receptor for human C5a anaphylatoxin. Nature 349:614. References 29. Boulay, F., L. Mery, M. Tardif, L. Brouchon, and P. Vignais. 1991. Expression 1. Johnston, R. B. 1988. Monocytes and macrophages. N. Engl. J. Med. 318:747. cloning of a receptor for C5a anaphylatoxin on differentiated HL-60 cells. Bio- 2. Goerdt, S., and C. E. Orfanos. 1999. Other functions, other genes: alternative chemistry 30:2993. activation of antigen-presenting cells. Immunity 10:137. 30. Ames, R. S., H. M. Sarau, P. Nuthulaganti, J. J. Foley, C. Ellis, Z. Zeng, K. Su, by guest on September 29, 2021 3. Becker, S., and E. G. Daniel. 1990. Antagonistic effects of IL-4 and interferon-␥ A. J. Jurewicz, R. P. Hertzberg, D. J. Bergsma, et al. 1996. Molecular cloning and on human monocytes and macrophages: effects on Fc receptors, HLA-D , characterization of the human anaphylatoxin C3a receptor. J. Biol. Chem. 271: and superoxide production. Cell. Immunol. 129:351. 20231. 4. Stein, M., S. Keshav, N. Harris, and S. Gordon. 1992. Interleukin-4 potently 31. Crass, T., U. Raffetseder, U. Martin, A. Grove, J. Klos, J. Ko¬hl, and W. Bautsch. enhances murine macrophage mannose receptor activity: a marker of alternative 1996. Expression cloning of the human C3a anaphylatoxin receptor (C3aR) from immunologic macrophage activation. J. Exp. Med. 176:287. differentiated U-937 cells. Eur. J. Immunol. 26:1944. 5. Hart, P. H., G. F. Vitti, D. R. Burgess, G. A. Whitty, D. S. Piccoli, and 32. Martin, U., D. Bock, L. Arseniev, M. A. Tornetta, R. S. Ames, W. Bautsch, J. J. A. Hamilton. 1989. Potential antiinflammatory effects of interleukin-4: sup- Ko¬hl, A. Ganser, and A. Klos. 1997. The human C3a receptor is expressed on pression of human monocyte tumor necrosis factor-␣, interleukin-1, and prosta- neutrophils and monocytes, but not on B or T lymphocytes. J. Exp. Med. 186:199. 33. Zwirner, J., O. Go¬tze, G. Begemann, A. Kapp, K. Kirchhoff, and T. Werfel. 1999. glandin E2. Proc. Natl. Acad. Sci. USA 86:3803. 6. Cheung, D. L., P. H. Hart, G. F. Vitti, G. A. Whitty, and J. A. Hamilton. 1990. Evaluation of C3a receptor expression on human leucocytes by the use of novel Contrasting effects of interferon-␥ and interleukin-4 on the interleukin-6 activity monoclonal . Immunology 97:166. of stimulated human monocytes. Immunology 71:70. 34. Nataf, S., N. Davoust, R. S. Ames, and S. R. Barnum. 1999. Human T cells 7. Standiford, T. J., S. L. Kunkel, J. M. Liebler, M. D. Burdick, and R. M. Strieter. express the C5a receptor and are chemoattracted to C5a. J. Immunol. 162:4018. 1993. Macrophage inflammatory protein-1␣ expression in interstitial lung dis- 35. Werfel, T., K. Kirchhoff, M. Wittmann, G. Begemann, A. Kapp, F. Heidenreich, ease. J. Immunol. 151:2852. O. Gotze, and J. Zwirner. 2000. Activated human T lymphocytes express a func- 8. Fenton, M. J., J. A. Buras, and R. P. Donnelly. 1992. IL-4 reciprocally regulated tional C3a receptor. J. Immunol. 165:6599. IL-1 and IL-1 receptor antagonist expression in human monocytes. J. Immunol. 36. Hartmann, K., B. M. Henz, S. Kru¬ger-Krasagakes, J. Ko¬hl, R. Burger, S. Guhl, 149:1283. I. Haase, U. Lippert, and T. Zuberbier. 1997. C3a and C5a stimulate chemotaxis 9. Schebesch, C., V. Kodelja, C. Mu¬ller, N. Hakij, C. E. Orfanos, and S. Goerdt. of human mast cells. Blood 89:2863. 1997. Alternatively activated macrophages actively inhibit proliferation of pe- 37. Daffern, P. J., P. H. Pfeifer, J. A. Ember, and T. E. Hugli. 1995. C3a is a che- ripheral blood lymphocytes and CD4ϩ T cells in vitro. Immunology 92:478. motaxin for human eosinophils but not for neutrophils. J. Exp. Med. 181:2119. 10. Sallusto, F., and A. Lanzavecchia. 1994. Efficient presentation of soluble antigen 38. Rothermel, E., O. Rolf, O. Go¬tze, and J. Zwirner. 1997. Nucleotide and corrected by cultured human dendritic cells is maintained by granulocyte/macrophage col- amino acid sequence of the functional recombinant rat anaphylatoxin C5a. Bio- ony-stimulating factor plus interleukin-4 and downregulated by tumor necrosis chim. Biophys. Acta 1351:9. factor ␣. J. Exp. Med. 179:1109. 39. Zwirner, J., O. Go¬tze, A. Moser., A. Sieber, G. Begemann, A. Kapp, J. Elsner, 11. Romani, N., S. Gruner, D. Brang, E. Kampgen, A. Lenz, B. Trockenbacher, and T. Werfel. 1997. Blood- and skin-derived monocytes/macrophages respond G. Konwalinka, P. O. Fritsch, R. M. Steinman, and G. Schuler. 1994. Prolifer- to C3a but not to C3a(desArg) with a transient release of calcium via a pertussis ating dendritic cell progenitors in human blood. J. Exp. Med. 180:83. toxin-sensitive signal transduction pathway. Eur. J. Immunol. 27:2317. 12. Banchereau, J., B. Schuler-Thurner, A. K. Palucka, and G. Schuler. 2001. Den- 40. Wilken, H. C., O. Gotze, T. Werfel, and J. Zwirner. 1999. C3a(desArg) does not dritic cells as vectors for therapy. Cell 106:271. bind to and signal through the human C3a receptor. Immunol. Lett. 67:141. 13. Lu, L., D. McCaslin, T. E. Starzl, and A. W. Thomson. 1995. Bone marrow 41. Heller, T., M. Hennecke, U. Baumann, J. E. Gessner, A. M. zu Vilsendorf, derived dendritic cell progenitors (NLDC 145ϩ, MHC class IIϩ, B7-1dim,B7Ð M. Baensch, F. Boulay, A. Kola, A. Klos, W. Bautsch, et al. 1999. Selection of 2Ϫ) induce alloantigen-specific hyporesponsiveness in murine T lymphocytes. a C5a receptor antagonist from phage libraries attenuating the inflammatory re- Transplantation 60:1539. sponse in disease and ischemia/reperfusion injury. J. Immunol. 14. Labeur, M. S., B. Roters, B. Pers, A. Mehling, T. A. Luger, T. Schwarz, and 163:985. S. Grabbe. 1999. Generation of tumor immunity by bone marrow-derived den- 42. Nakajima-Iijima, S., H. Hamada, P. Reddy, and T. Kakunaga. 1985. Molecular dritic cells correlates with dendritic cell maturation stage. J. Immunol. 162:168. structure of the human cytoplasmic ␤-actin gene: interspecies homology of se- 15. Inaba, K., M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. Muramatsu, quences in the introns. Proc. Natl. Acad. Sci. USA 82:6133. and R. M. Steinman. 1992. Generation of large numbers of dendritic cells from 43. Roglic, A., E. R. Prossnitz, S. L. Cavanagh, Z. Pan, A. Zou, and R. D. Ye. 1996. mouse bone marrow cultures supplemented with granulocyte/macrophage colo- cDNA cloning of a novel G protein-coupled receptor with a large extracellular ny-stimulating factor. J. Exp. Med. 176:1693. loop structure. Biochim. Biophys. Acta 1305:39. 3314 IL-4 DOWN-REGULATES ANAPHYLATOXIN RECEPTORS

44. Tokunaga, K., H. Taniguchi, H. Yoda, M. Shimizu, and S. Sakiyama. 1986. 55. Bonecchi, R., F. Facchetti, S. Dusi, W. Luini, D. Lissandrini, M. Simmelink, Nucleotide sequence of a full-length cDNA for mouse cytoskeletal ␤-actin M. Locati, S. Bernasconi, P. Allavena, E. Brandt, et al. 2000. Induction of func- mRNA. Nucleic Acids Res. 14:2829. tional IL-8 receptors by IL-4 and IL-13 in human monocytes. J. Immunol. 164: 45. Gerard, C., L. Bao, O. Orozco, M. Pearson, D. Kunz, and N. P. Gerard. 1992. 3862. Structural diversity in the extracellular faces of peptidergic G-protein-coupled 56. Karp, C. L., A. Grupe, E. Schadt, S. L. Ewart, M. Keane-Moore, P. J. Cuomo, receptors. J. Immunol. 149:2600. J. Kohl, L. Wahl, D. Kuperman, S. Germer, et al. 2000. Identification of com- 46. Marder, S. R., D. E. Chenoweth, I. M. Goldstein, and H. D. Perez. 1985. Che- plement factor 5 as a susceptibility locus for experimental allergic asthma. Nat. motactic responses of human peripheral blood monocytes to the complement- Immunol. 1:221. derived peptides C5a and C5adesArg. J. Immunol. 134:3325. 57. Wittmann, M., J. Zwirner, V. A. Larsson, K. Kirchhoff, G. Begemann, A. Kapp, 47. Chenoweth, D. E., M. G. Goodman, and W. O. Weigle. 1982. Demonstration of O. Gotze, and T. Werfel. 1999. C5a suppresses the production of IL-12 by IFN- a specific receptor for human C5a anaphylatoxin on murine macrophages. J. Exp. ␥-primed and lipopolysaccharide-challenged human monocytes. J. Immunol. Med. 156:68. 162:6763. 48. Zwirner, J., T. Werfel, H. C. Wilken, E. Theile, and O. Go¬tze. 1998. Anaphyla- 58. Braun, M. C., E. Lahey, and B. L. Kelsall. 2000. Selective suppression of IL-12 toxin C3a but not C3a(desArg) is a chemotaxin for the mouse macrophage cell production by chemoattractants. J. Immunol. 164:3009. line J774. Eur. J. Immunol. 28:1570. 59. Cavaillon, J. M., C. Fitting, and N. Haeffner-Cavaillon. 1990. Recombinant C5a 49. Wilkinson, P. C. 1996. Cell locomotion and chemotaxis: basic concepts and enhances interleukin 1 and tumor necrosis factor release by lipopolysaccharide- methodological approaches. Methods 10:74. stimulated monocytes and macrophages. Eur. J. Immunol. 20:253. 50. Dunzendorfer, S., A. Kaser, C. Meierhofer, H. Tilg, and C. J. Wiedermann. 2000. Dendritic cell migration in different micropore filter assays. Immunol. Lett. 71:5. 60. Scholz, W., M. R. McClurg, G. J. Cardenas, M. Smith, D. J. Noonan, T. E. Hugli, 51. Hugli, T. E. 1990. Structure and function of C3a anaphylatoxin. Curr. Top. Mi- and E. L. Morgan. 1990. C5a-mediated release of interleukin 6 by human mono- crobiol. Immunol. 153:181. cytes. Clin. Immunol. Immunopathol. 57:297. 52. Naik, N., E. Giannini, L. Brouchon, and F. Boulay. 1997. Internalization and 61. Ember, S. D. Sanderson, T. E. Hugli, and E. L. Morgan. 1994. Induction of recycling of the C5a anaphylatoxin receptor: evidence that the agonist-mediated interleukin-8 synthesis from monocytes by human C5a anaphylatoxin. internalization is modulated by phosphorylation of the C-terminal domain. J. Cell Am. J. Pathol. 144:393. Sci. 110:2381. 62. McWilliam, A. S., D. Nelson, J. A. Thomas, and P. G. Holt. 1994. Rapid den- 53. Settmacher, B., D. Bock, H. Saad, S. Gartner, C. Rheinheimer, J. Kohl, dritic cell recruitment is a hallmark of the acute inflammatory response at mu- Downloaded from W. Bautsch, and A. Klos. 1999. Modulation of C3a activity: internalization of the cosal surfaces. J. Exp. Med. 179:1331. human C3a receptor and its inhibition by C5a. J. Immunol. 162:7409. 63. Scha¬kel K., R. Kannagi, B. Kniep, Y. Goto, C. Mitsuoka, J. Zwirner, A. Soruri, 54. Wang, J., L. Wang, J. Zheng, J. L. Anderson, and M. L. Toews. 2000. Identifi- M. v. Kietzell, and E. P. Rieber. 2002. 6-SulfoLacNAc, a novel carbohydrate cation of distinct carboxyl-terminal domains mediating internalization and down- modification of PSGL-1, defines an inflammatory type of human dendritic cells. regulation of the hamster ␣(1B)-adrenergic receptor. Mol. Pharmacol. 57:687. Immunity 17:289. http://www.jimmunol.org/ by guest on September 29, 2021