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The Journal of Immunology, 2003, 170: 3306-3314.
Copyright © 2003 by The American Association of Immunologists

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{dagger}, Jörg Köhl{ddagger} and Jörg Zwirner2,*

Departments of * Immunology and {dagger} Transfusion Medicine, Georg August University Gottingen, Gottingen, Germany; and {ddagger} Department of Molecular Immunology, Childrens Hospital Research Foundation, Cincinnati, OH 45229


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Anaphylatoxins mobilize leukocytes to the sites of inflammation. In the present study we investigated the impact of GM-CSF, IL-4, and IFN-{gamma} 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 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Macrophages may be activated by the Th1 cytokine IFN-{gamma}, resulting in up-regulation of MHC class II and Fc{gamma}Rs and increased production of proinflammatory cytokines such as IL-1, IL-6, TNF-{alpha}, and chemokines (1, 2). IL-4, a Th2 cytokine, is a counterplayer of IFN-{gamma} (3). Its effects on macrophages have been described as alternative activation (4), which includes induction of MHC class II and mannose receptor expression, but also inhibition of proinflammatory cytokine secretion (e.g., IL-1, TNF-{alpha}, IL-6, and IL-12) (5, 6, 7). With respect to down-regulation of inflammation, alternatively activated macrophages are characterized by expression of anti-inflammatory cytokines such as IL-10 and IL-1 receptor antagonist (8, 9).

IL-4 is instrumental, in combination with GM-CSF, for the transition of monocytes into dendritic cells (DC)3 in vitro (10, 11). Monocyte-derived DC (MoDC) generated under these conditions are regarded as equivalent to immature DC and are widely used for experimental as well as clinical purposes (12). IL-4 has also been used for the generation of CD34+ cell-derived DC by some investigators (13, 14), but not by others (15, 16).

The main function of DC is to collect Ags in inflamed tissues and to migrate to the local lymph nodes, where specific immune responses are initiated (17). Migration of DC is critically governed by the differential expression of chemokine receptors (18). Immature DC are responsive to inflammatory chemokines (19, 20), which may guide them to inflammatory sites where Ag sampling can take place, and maturation is induced. The maturation process, which may be triggered by inflammatory stimuli such as IL-1{beta}, TNF-{alpha}, LPS, or CD40 ligand (CD40L) (10, 21) leads to down-regulation of receptors for inflammatory and up-regulation of receptors for constitutive chemokines, such as macrophage inflammatory protein 3{beta} (MIP-3{beta}), which may induce migration of DC to lymphoid organs (22, 23). As a classical inflammatory stimulus, the anaphylatoxin C5a has been shown to be a chemoattractant for immature DC (19). However, controversial results exist regarding the reactivity of mature DC toward C5a (24, 25).

Anaphylatoxins are generated by activation-induced cleavage of the third and fifth components of complement. C5a and, to a lesser extent, C3a are mediators of proinflammatory and immunoregulatory activities (26, 27). The C3a (77aa) and C5a (74aa) peptides regulate inflammatory functions by interacting with their receptors, C3aR and C5aR, both of which belong to the rhodopsin family of seven-transmembrane, G protein-coupled receptors (28, 29, 30, 31). Anaphylatoxin receptors are present on myeloid and nonmyeloid leukocyte populations, including granulocytes and monocytes/macrophages (32, 33), lymphocytes (34, 35), and DC (19, 24). Whereas C5a is a potent chemotaxin for all C5aR-expressing cell types, C3a-induced mobilization of primary cells has only been demonstrated for eosinophils and mast cells (36, 37).

The purpose of the present study was to evaluate anaphylatoxin-induced mobilization of monocytes and DC in an in vivo SCID mouse model and to investigate the impact of IL-4 on anaphylatoxin receptor expression and function. Our results confirmed C5a and newly established C3a as chemoattractants for monocytes/macrophages. IL-4 was found to down-regulate anaphylatoxin receptors in human monocytes and MoDC as well as in murine bone marrow-derived DC (BmDC). In parallel with receptor down-regulation, anaphylatoxin-induced mobilization was inhibited.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Recombinant chemotaxins

Recombinant human anaphylatoxins C5a (38), C3a (39), and C3a(desArg) (40) were generated as described. LPS concentrations, determined by the Limulus assay (Coatest Endotoxin; Pharmacia, Freiburg, Germany), were <20 pg LPS/µg anaphylatoxin. Recombinant C5aR antagonist (C5aRA) was generated as previously described (41). MIP-1{alpha} and MIP-3{beta} were obtained from PeproTech (Cell Concepts, Umkirch, Germany).

Monoclonal Ab against anaphylatoxin receptors

The mAb hC3aRZ8 (IgG2b) was generated by immunizing BALB/c mice i.p. with RBL-2H3 transfectants expressing the human C3aR and fusion of spleen cells with the myeloma cell line X63-Ag8.653. Supernatants of hybridomas generated according to standard techniques were tested for their reactivity with a recombinant C3aR fragment of the large extracellular loop structure of the human C3aR and with human C3aR expressing RBL-2H3 transfectants. Immunofluorescent staining obtained with mAb hC3aRZ8 and mAbs anti-C3aR hC3aRZ1-4, which have been previously generated by our laboratory (33), were similar (not shown).

The mAb hC5aRZ1 (IgG1) was generated by immunizing BALB/c mice with RBL-2H3 transfectants expressing the human C5aR. Supernatants of hybridomas were tested for their reactivity with peptides representing the N-terminal 31 aa of the C5aR and with C5aR expressing RBL-2H3 transfectants. In additional experiments mAb C5aRZ1 was shown to block binding of fluorescein-conjugated recombinant C5a to its receptor on RBL-2H3 transfectants.

Human monocytes/macrophages and MoDC

Leukocytes were obtained by leukapheresis from volunteer blood donors at University Clinic Gottingen. PBMC were isolated by centrifugation on a Ficoll-Hypaque discontinuous gradient. PBMC were cultured for 1 h at 1 x 107 cells/ml in endotoxin-free RPMI 1640 (Biochrom, Berlin, Germany) supplemented with 5% heat-inactivated autologous serum in flat-bottom plates. After washing off nonadherent cells, adherent PBMC (>80% monocytes) were cultured in RPMI 1640 supplemented with 10% FCS (PAN Biotech, Aidenbach, Germany), penicillin/streptomycin, L-glutamine, GM-CSF (300 U/ml), and IL-4 (300 U/ml; both from R&D Systems, Wiesbaden, Germany). After 5–7 days, cultured MoDC expressed HLA-DR, but not CD14, and were characterized as immature due to their moderate expression of CD86 and low expression of CD83. Further differentiation into mature DC was accomplished by incubation with CD40L/enhancer (100 ng/ml; Alexis, San Diego, CA) plus TNF-{alpha} (25 ng/ml; R&D Systems) for 48 h.

To investigate the effects of saturating concentrations of GM-CSF (300 U/ml), IL-4 (300 U/ml), and IFN-{gamma} (300 U/ml; all from R&D Systems) on anaphylatoxin receptor expression in human monocytes, adherent PBMC were cultured in RPMI with 10% FCS for 2 days. To investigate the effect of IL-4 on MoDC, adherent PBMC were cultured in RPMI 1640 with 10% FCS in the presence of saturating concentrations of GM-CSF (300 U/ml) with or without IL-4 (300 U/ml) for 5 days. To obtain macrophages, adherent PBMC were cultured in the presence of 5% pooled human serum for 2 or 4 days.

Murine BmDC

A modified method of Inaba et al. (15) for the isolation of bone marrow precursors was used. Briefly, bone marrow was collected from tibias and femurs of female BALB/c mice, passed through a nylon mesh to remove small pieces of bone and debris, resuspended in complete medium (CM: RPMI 1640 containing 5% FCS, 20 µg/ml gentamicin, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 µM 2-ME), and cultured in tissue culture dishes for 2 h. Nonadherent cells were collected, and aliquots of 1 x 106 cells were placed in 24-well plates containing 1 ml of CM with two different combination of cytokines: 100 U/ml GM-CSF with or without 200 U/ml IL-4 (both from Cell Concepts). Two-thirds of the medium was replaced on days 3 and 5. On day 6 of culture, nonadherent cells were transferred into six-well plates in CM (1 x 106 cells/ml) with cytokines and maintained for 2 additional days. Both BmDC preparations were immature, as characterized by their low expression of MHC class II I-E, CD80, and CD86 and low stimulation of allogeneic T lymphocytes, but could be differentiated with LPS into mature, highly stimulatory DC (data not shown).

Flow cytometric analysis

For analysis of human Ags the following Abs were used: anti-HLA-DR (Hölzl Diagnostics, Koln, Germany), anti-CD86, anti-CD14 (both from Dakopatts, Hamburg, Germany), and anti-CD83 (Beckman Coulter, Krefeld, Germany). All Abs were either FITC- or PE-conjugated as indicated. Cells (2 x 105) were washed with PBS containing 1.5% FCS and 10 mM sodium azide. They were then blocked with heat-aggregated human IgG (and murine IgG if murine cells were stained; 20 µg each in 100 µl) for 20 min on ice. After washing three times with PBS containing 1.5% FCS and 10 mM sodium azide, cells were incubated with labeled Ab for 45 min. As a negative control, FITC-conjugated IgG1/PE-conjugated IgG2a murine isotype mix was used (Dakopatts). Finally, cells were washed as described above, resuspended in PBS containing 1% formaldehyde, and analyzed in a flow cytometer (EPICS XL; Beckman Coulter, Krefeld, Germany). Gating was performed to exclude dead cells from analyses.

If indirect immunofluorescence staining was performed, the following Abs were used: mAb anti-C5aR (hC5aRZ1), mAb anti-C3aR (hC3aRZ8), irrelevant control mAb, and goat anti-mouse IgG (H+L) FITC-conjugated (Dakopatts).

Migration of human cells in an SCID mouse model

All animal work was conducted in accordance with guidelines for the welfare of animals and was approved by the administration of Lower Saxony, Germany. SCID mice (strain CB-17 scid of both sexes; 19–24 g) were obtained from the University of Gottingen or from Charles River (Sulzfeld, Germany). Human cells (1 x 107) were resuspended in 200 µl of PBS and injected into the tail vein of SCID mice. In parallel, 200 µl of PBS containing recombinant anaphylatoxin (2.5–40 µg) was injected i.p. After 2, 4, 24, or 48 h, mice were sacrificed, and cells were harvested from the peritoneum by flushing with 7 ml of PBS and counted using a hemocytometer. Thereafter, migrated human monocytes, macrophages, or MoDC were 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 flow cytometry. Absolute numbers of migrated human cells were calculated from the percentage of HLA-DR+ cells and the total peritoneal cell count. Human or murine B (CD19+) and T (CD3+) lymphocytes were never detectable in the peritoneal cavity of SCID mice.

To block migration, human cells (1 x 107) were incubated with 50 µg of mAb (anti-C5aR hC5aRZ1, anti-C3aR hC3aRZ8) in 1 ml of RPMI for 1 h on ice. Cells were washed once, transferred into PBS, and injected i.v.

Migration of murine BmDC in vivo

Murine BmDC were labeled with the red fluorescent dye PKH-26 (Sigma-Aldrich, Deisenhofen, Germany) according to the manufacturer's instructions. Labeled BmDC (1 x 107 in 200 µl of PBS) were injected into the tail vein of BALB/c mice (weight, 25–29 g; age, 8–20 wk). Immediately thereafter, the chemotaxin (10 µg in 200 µl; in some experiments 5–40 µg as indicated) was injected into the peritoneal cavity. Two to 48 h later, mice were killed, and peritoneal lavage was performed using 10 ml of PBS. Subsequently, peritoneal cells were counted using a hemocytometer and analyzed by FACS. Absolute numbers of migrated BmDC were calculated from the percentage of red fluorescent cells and the total peritoneal cell count. Red-labeled granulocytes or lymphocytes that might contaminate BmDC preparations to some degree were never observed in the peritoneal cavity of injected BALB/c mice.

RT-PCR

Total RNA was isolated using a commercially available preparation method (NucleoSpin; Macherey-Nagel, Duren, Germany). RNA was digested with DNase I to exclude genomic DNA contamination. First-strand cDNA synthesis on 2–5 µg of RNA was performed using the SuperScript Preamplification Kit (Life Technologies, Eggenstein, Germany) with random hexanucleotide primers. The PCR mixture (50 µl) contained varying amounts of cDNA, 0.2 mM dNTP, 0.5 µM 5' and 3' oligonucleotide primers as well as reaction buffer and 1.25 U of DNA polymerase (HotStarTaq; Qiagen, Hilden, Germany). DNA amplification was conducted using a thermocycler (Mastercycler; Eppendorf, Hamburg, Germany). PCR conditions were 28 cycles of 94°C (1 min), 59°C (1 min), and 72°C (1 min) for human {beta}-actin; 32 cycles of 94°C (1 min), 59°C (1 min), and 72°C (1 min) for human C5a and C3a receptors; 28 cycles of 94°C (1 min), 50°C (1 min), and 72°C (1 min) for murine {beta}-actin; and 27 cycles of 94°C (1 min), 67°C (1 min), and 72°C (1 min) for murine C5aR. The following oligonucleotide primer sets were obtained from MWG-Biotech (Edersberg, Germany): human {beta}-actin: sense, 5'-AAGGCCAACCGCGAGAAGATGA; and antisense, 5'-GGAAGAGTGCCTCAGGGCAGCG (amplifying a 451-bp fragment) (42); human C5aR: sense, 5'-CAGGAGACCAGAACATGAACTCC; and antisense, 5'-TACATGTTGAGCAGGATGAGGG (ampli-fying a 376-bp fragment) (29); human C3aR: sense, 5'-CAGACAGGACTCGTGGAGAC; and antisense, 5'-GACAATGATGGAGGGGATGAG (amplifying a 381-bp fragment) (43); murine {beta}-actin: sense, 5'-TGGAATCCTGTGGCATCCATGAAAC; and antisense, 5'-TAAAACGCAGCTCAGTAACAGTCCG (amplifying a 348-bp fragment) (44); murine C5aR: sense, 5'-AAGGTCCGCGGGACTGGCCTGG; and antisense, 5'-GAGAGCGTTTCGTATGATGCTGGGG (amplifying a 536-bp fragment) (45). PCR products (10 µl) were separated by electrophoresis in 1.2–1.5% agarose gels and visualized by UV light illumination after ethidium bromide staining. To control for saturation effects of the PCR reaction, preliminary experiments were assayed at different cycle numbers by removing part of the reaction at appropriate times.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Anaphylatoxin receptor expression in human monocytes and DC

GM-CSF and IL-4 are commonly used for the generation of DC from monocytes. We therefore investigated the impact of GM-CSF and IL-4 compared with IFN-{gamma} on C5aR and C3aR expression in monocytes after a 2-day incubation period. The strongest down-regulation of C5aR and C3aR proteins as detected by mAbs was seen for IL-4 and IL-4 plus GM-CSF, whereas GM-CSF and IFN-{gamma} alone were less potent (Fig. 1). IL-4 also down-regulated C5aR mRNA, as documented by RT-PCR (Fig. 1). The negative impact of IL-4 on C5aR number was independent of the source of serum used, as it was also observed in the presence of human serum (Fig. 1). Table I shows the impact of IL-4 on anaphylatoxin receptor expression over time.



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  		FIGURE 1. Regulation of C5aR and C3aR expression in monocytes. Monocytes were cultured for 2 days in FCS (A and C) or human serum (B) in the presence or the absence (control) of cytokines. Cells were analyzed for the expression of C5aR and C3aR by indirect immunofluorescence staining and FACS. The mean ± SE of at least five independent experiments are shown. D, RT-PCR analysis of C5aR and {beta}-actin mRNA in monocytes cultured for 2 days in the presence and the absence of IL-4 is demonstrated.

 

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  		Table I. C5aR and C3aR expression in monocytes over timea

 
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.



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  		FIGURE 2. IL-4 down-regulates anaphylatoxin receptor expression in MoDC. Monocytes were cultured for 5 days in GM-CSF with or without IL-4 and were analyzed by FACS. A, Dot plots show double-immunolabeled cells. B, Histograms show cells stained with mAbs against C3aR and C5aR by indirect immunofluorescence. Data are representative of three independent experiments. C, RT-PCR analysis of {beta}-actin, C3aR, and C5aR in monocytes cultured for 5 days in GM-CSF with or without IL-4 is shown.

 
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 isolated human monocytes or MoDC and i.p. injection of C5a (10 µg), 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.


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  		Table II. Peritoneal mobilization of human monocytes and MoDC to C5aa

 
We then explored the impact of increasing amounts of C5a on the peritoneal influx of monocytes or macrophages 4 and 24 h after their injection (Fig. 3). Migration of these cells was inducible by 5 and 10 µg of C5a at both time points investigated, whereas 2.5 µg was ineffective. C3a also recruited human monocytes/macrophages into the peritoneal cavity; however, distinctly higher quantities, compared with those of C5a, were required. The C-terminally desarginated catabolite of C3a, C3a(desArg), was completely ineffective as a chemoattractant for monocytes/macrophages using up to 40 µg/animal. Furthermore, monocytes/macrophages did not migrate i.p. if PBS was injected (n = 6; data not shown).



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  		FIGURE 3. Dose-dependent migration 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 received C5a (2.5, 5, or 10 µg) or C3a (10, 20, or 40 µg) i.p. 4 or 24 h later, peritoneal cells were harvested, and human cells were analyzed by FACS after staining with PE-conjugated anti-HLA-DR and FITC-conjugated anti-CD14. A, Absolute numbers of migrated HLA-DR+ human cells were calculated from the percentage of red fluorescent cells and the total peritoneal cell count; B, dot plot analyses of double-labeled human cells are shown.

 
We also investigated whether C5a-mediated recruitment in vivo was dependent on signaling through the C5aR. When human monocytes were preincubated in vitro with anti-C5aR mAb specifically blocking the binding of C5a to human C5aR, their mobilization was abrogated (Table III). However, binding of anti-C5aR mAb did not impede the ability of monocytes to migrate in response to MIP-1{alpha}. Furthermore, preincubation with anti-C3aR mAb was without influence on C5a-induced peritoneal influx of monocytes. Thus, the possibility was excluded that anti-C5aR mAb blocks cell migration unspecifically. These control experiments also demonstrate that the C5a does not exert its chemotactic effects by inducing the release of secondary chemotactic substances from resident peritoneal cells.


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

 
Reagents to specifically block C3aR-mediated activities are not available. To inhibit chemotaxis of monocytes in a C3aR-specific manner, we took advantage of the reported internalization of anaphylatoxin receptors upon ligand exposure. Preincubation of monocytes overnight with C3a completely blocked their C3a-induced migration 4 and 24 h after injection, whereas C5a-induced migration was unaffected (Fig. 4A). Vice versa, if monocytes were preincubated with C5a, C5a-induced, but not C3a-induced, migration was inhibited. Preincubation of monocytes with pertussis toxin also abolished migration toward anaphylatoxins in vivo, which confirmed the involvement of G proteins in signal transduction (Fig. 4B).



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  		FIGURE 4. Preincubation with anaphylatoxins and pertussis toxin blocks C5a- and C3a-mediated migration of macrophages in vivo. Macrophages (cultured for 4 days in 5% human serum) were incubated overnight in the presence or the absence of anaphylatoxin (2 µg/ml; A) or pertussis toxin (100 ng/ml; B). Cells were then injected into SCID mice i.v. that at the same time received C3a or C5a i.p. After 24 h, peritoneal cells were harvested, counted, and stained with PE-anti-HLA-DR. Absolute numbers of migrated HLA-DR+ cells were calculated from the percentage of red fluorescent cells determined by FACS analysis and the total peritoneal cell count. The mean values of two independent experiments are shown.

 
Impact of IL-4 on anaphylatoxin-induced in vivo migration of monocytes and DC

We investigated the functional consequences of IL-4-induced C5aR and C3aR down-regulation in monocytes in the SCID mouse model. IL-4 treatment abolished cell migration in response to C5a (10 µg) or C3a (40 µg) as measured 4 h after their injection (Fig. 5A). Unresponsiveness of IL-4-treated monocytes could be overcome by injecting 20 µg of C5a (Fig. 5B). Migration in response to 20 µg of C5a was ligand-specific, as it could be abolished by C5aR blockade.



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  		FIGURE 5. IL-4 inhibits anaphylatoxin-mediated migration of monocytes. A, Monocytes were cultured for 2 days in the presence or the absence of IL-4. Cells were then injected into SCID mice i.v. that at the same time received C3a or C5a i.p. After 4 h, peritoneal cells were harvested, counted, and stained with PE-anti-HLA-DR. Absolute numbers of migrated HLA-DR+ cells were calculated from the percentage of red fluorescent cells as analyzed by FACS and the total peritoneal cell count. The mean ± SE of three independent experiments are shown. B, Migration of IL-4-treated monocytes in response to 20 µg of C5a in vivo could be blocked by preincubation with mAb anti-C5aR hC5aRZ1.

 
IL-4 also delayed the migration of MoDC in response to anaphylatoxins (Fig. 6). Following peritoneal C5a (10 µg) injection, MoDC were undetectable after 4 h, whereas after 24 h, migration was evident (Fig. 6A). Increasing the amount of C5a to 20 µg partially restored the responsiveness of MoDC, as cells were detectable i.p. even 4 h after their injection. In contrast to C5a, C3a up to 40 µg was not effective in mobilizing MoDC into the peritoneal cavity of SCID mice. Fig. 6B demonstrates that it was IL-4 that impaired anaphylatoxin-induced mobilization of MoDC in vivo. Whereas 5 µg 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 abolished the reactivity of MoDC to C3a (Fig. 6B).



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  		FIGURE 6. IL-4 inhibits migration of MoDC. A, MoDC cultured in GM-CSF and IL-4 were injected i.v. into SCID mice. After 4 or 24 h, peritoneal cells were harvested, counted, and stained with PE-anti-HLA-DR. The mean ± SE of at least four independent experiments are shown. B, After culturing monocytes for 5 days with GM-CSF or GM-CSF plus IL-4 (MoDC), cells were injected into SCID mice i.v., which at the same 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 independent experiments are shown. Absolute numbers of migrated HLA-DR+ cells were calculated from the percentage of red fluorescent cells as determined by FACS and the total peritoneal cell count.

 
Mobilization of injected MoDC was ligand specific, as it was inhibitable by C5aR blockade (Table III). Furthermore, no migration of MoDC was seen if PBS was injected (n = 3; data not shown).

Impact of maturation on the in vivo migration of DC

Incubation of MoDC in the presence of CD40L and TNF-{alpha} resulted in phenotypic and functional maturation, as evidenced by the strong up-regulation of CD83, CD86, and HLA-DR and the 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 µg) injection (Fig. 7). Instead, mature MoDC gained responsiveness to MIP-3{beta}. Migration of monocytes (n = 4) and immature MoDC (n = 4) in response to MIP-3{beta} (10 µg) was not observed (data not shown).



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  		FIGURE 7. Mature MoDC gain responsiveness to MIP-3{beta}. Mature MoDC were generated by culturing monocytes for 5 days with GM-CSF and IL-4 and for 2 additional days with TNF-{alpha} and CD40L. Cells were injected i.v. into SCID mice that at the same time received C5a (10 µg) or MIP-3{beta} (10 µg) i.p. 24 h later, peritoneal cells were harvested and human cells were identified by PE-anti-HLA-DR in combination with FITC-anti-CD86 or FITC-anti-CD83. One representative of four independent experiments is shown.

 
In vivo migration of BmDC in syngeneic BALB/c mice

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).



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  		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 x 107) together with 10 µg 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 numbers of migrated BmDC were calculated from the percentage 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.

 
Migration of BmDC to C5a was dependent on its interaction with the C5aR. Preincubation of BmDC with C5aRA, which specifically 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

Murine BmDC lost their ability to migrate in response to C5a (10 µg) in vivo when they were cultured with IL-4. Neither 4 nor 24 h after the injection of C5a were IL-4-treated BmDC detectable in the peritoneal cavity, with the exception of one experiment in which 9200 labeled cells were counted (Fig. 9A). Parallel to its negative impact on C5aR-mediated chemotaxis, IL-4 treatment also down-modulated the expression of C5aR mRNA in BmDC (Fig. 9B).



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  		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 injected i.v. (1 x 107) together with 10 µg of C5a i.p. into BALB/c mice. Peritoneal cells were harvested 4 or 24 h later, counted, and analyzed by FACS. Absolute numbers of migrated BmDC were calculated from the percentage of migrated labeled cells and the total peritoneal cell count. The mean ± SE of at least six independent experiments are shown. B, RT-PCR results for {beta}-actin and C5aR in BmDC cultured in the absence or the presence of IL-4 are shown.

 
We also examined whether higher doses of C5a could overcome the unresponsiveness of IL-4-treated BmDC toward the anaphylatoxin C5a in vivo. Fig. 10 demonstrates that this was indeed the case. When IL-4-treated BmDC were harvested 4 h after injection, 40 µg of C5a were required to induce a peritoneal influx of labeled BmDC. After 24 h, 20 µg of C5a were required to induced the migration of BmDC. In contrast, peritoneal mobilization of BmDC that were cultured in the presence of GM-CSF alone was observed with as little as 5 µg of C5a after 4 h.



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  		FIGURE 10. Increasing amounts of C5a overcome IL-4-induced unresponsiveness of murine BmDC to C5a in vivo. GM-CSF-treated and GM-CSF- plus IL-4 treated BmDC were labeled with the red fluorescent dye PKH-26 and injected i.v. (1 x 107 cells) together with varying amounts of C5a i.p. into BALB/c mice. Peritoneal cells were collected 4 or 24 h after C5a injection, counted, and analyzed by FACS. Absolute numbers of migrated BmDC were calculated from the percentage of red fluorescent cells and the total peritoneal cell count.

 
Peritoneal influx of BmDC (treated with GM-CSF with or without IL-4) was never observed in the absence of anaphylatoxin challenge (n = 16), excluding the possibility that BmDC spontaneously migrated i.p.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
One of the main functions of anaphylatoxins is the recruitment of leukocytes to the sites of infection, inflammation, and trauma. Chemotactic responses of monocytes and macrophages to C5a have been described in the past (46, 47). However, no such report exists for C3a, and only the murine macrophage cell line J774A.1 could be mobilized by C3a gradients in vitro (48). This may not be surprising, as primary monocytes express ~6 times more C5aR than C3aR molecules on their surface (33). Our laboratory has been unable to detect directed migration of monocytes/macrophages to C3a using a standard in vitro microchemotaxis assay and polycarbonate membranes (J. Zwirner, unpublished observations). This in vitro approach has been shown to be hampered by a lack of sensitivity and may not permit discrimination between chemotactic and chemokinetic cell motilities, since cell velocities cannot be calculated (49, 50).

Therefore, in the present study we investigated migration in vivo using immunodeficient SCID mice as recipients of human cells. Human monocytes injected i.v. accumulated in the peritoneal cavity if C3a was present. Compared with C5a, higher amounts of C3a had to be injected to attract similar cell numbers. This observation can be explained by differences in absolute anaphylatoxin receptor numbers on monocytes (33) and by carboxypeptidase-mediated removal of the terminal arginine in vivo, which completely inactivates C3a, but not C5a (51). It was therefore not surprising that the catabolite of C3a, C3a(desArg), was ineffective in attracting monocytes/macrophages in vivo. The ubiquitous carboxypeptidase N may constitute an essential regulatory mechanism to control C3a activity in vivo, as there is 10 times more C3 than C5 present in serum (51). Of note, a spontaneous random influx of human monocytes/macrophages into the peritoneal cavity in the absence of anaphylatoxins did not occur. Therefore, anaphylatoxin-induced mobilization in the SCID mouse model appears to be exclusively chemotactic in nature.

Agonist-induced, rapid receptor desensitization and internalization are important control mechanisms described for G protein-coupled receptors, including C3aR and C5aR (52, 53). If agonist exposure is prolonged, longer-lasting down-regulation has been shown for some G protein-coupled receptors (54). We now demonstrate that prolonged exposure of monocytes to C3a inhibited migration in vivo in response to C3a for at least 24 h, whereas preincubation with C5a did not affect C3a-induced mobilization and vice versa. These data suggest a ligand-specific functional down-regulation of anaphylatoxin receptors. It may serve to limit excessive proinflammatory activities of anaphylatoxins during continuous complement activation in vivo.

IL-4-treatment down-regulated anaphylatoxin receptors in monocytes and inhibited their mobilization in response to anaphylatoxins in vivo. In contrast, IL-4 has been shown recently to up-regulate IL-8Rs (CXCR1 and CXCR2) in human monocytes, which suggests that IL-8 contributes to the accumulation and positioning of mononuclear phagocytes in Th2-dominated responses (55). Vice versa, the regulatory effect of IL-4 on C5aR expression may be linked to the down-regulation of Th1 responses. Indeed, blockade of C5aR rendered human monocytes unable to produce IL-12 (56). It is tempting to speculate that during Th2 responses, IL-4 prevents the recruitment of monocytes and the production of IL-12 through C5aR down-regulation. On the other hand, C5a has been shown to suppress IL-12 production in stimulated monocytes (57, 58). Additional experiments are needed to reconcile these conflicting results.

C5a is a potent proinflammatory stimulus that attracts and degranulates monocytes/macrophages as well as induces them to produce IL-1{beta}, IL-6, IL-8, and TNF-{alpha} (59, 60, 61). The proinflammatory activity of monocytes following exposure to C3a is less evident. Human monocytes responded to C3a with an increase in intracellular calcium ions (39). Furthermore, the murine macrophage cell line J774A.1 migrated to C3a gradients in vitro (48). Our results now demonstrate for the first time that a complex cellular response such as migration in vivo can be induced in monocytes via C3aR-mediated activation. Inhibition of C3a-induced, in addition to C5a-induced mobilization of monocytes in vivo may thus represent an important anti-inflammatory activity of IL-4.

IL-4 plays a central role in the generation of DC in vitro, in particular if monocytes are used as progenitor cells (10, 11). In the present study we observed that IL-4 is a negative regulator of numbers and function of anaphylatoxin receptors in MoDC. However, C5a-induced migration of MoDC has been documented by other groups using in vitro chemotaxis assays (19, 25). Our data confirm these results, as MoDC also migrated in response to C5a in vivo; however, IL-4 treatment considerably reduced the sensitivity of DC to C5a stimulation and abrogated their responsiveness to C3a. IL-4 is also included in protocols for the generation of DC from bone marrow progenitor cells, although its use may not be obligatory. As shown for MoDC, IL-4 exerted a negative regulatory 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 vitro do not represent the equivalent of inflammatory DC in vivo, as rapid influx of DC is a hallmark of the acute inflammatory response in vivo (62). This hypothesis is supported by our recent finding that M-DC8+, blood-derived, immature DC represent an inflammatory type of DC that responds to C5a with a prompt and robust mobilization in the SCID mouse model (63).

C5a has been suggested to be involved in the trafficking of immature as well as mature DC, as both cell types expressed equal numbers of C5aR and showed no differences in their ability to migrate to C5a in vitro (25). We now demonstrate that mature MoDC are further inhibited in their ability to migrate to C5a compared with immature DC, whereas they gained responsiveness to MIP-3{beta}. A possible explanation for the discrepant results may be that IL-4-treated DC already express drastically reduced C5aR numbers compared with DC cultured in the absence of IL-4. A further reduction of receptor numbers by DC maturation may be susceptible to minor variations in culture conditions and thus not always be measurable.

In summary, this study shows that C3a recruits monocytes/macrophages in a receptor-specific manner in vivo, although it is not as effective as C5a. Impairment of anaphylatoxin-induced mobilization of monocytes and DC by IL-4 correlates with down-regulation of anaphylatoxin receptors. We suggest that inhibition of a rapid recruitment of monocytes and DC represents an important anti-inflammatory activity of the Th2 cytokine IL-4.


   Acknowledgments
 
We thank Jutta Wollenweber and Ines Heine for their excellent technical assistance.


   Footnotes
 
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 für Klinische Forschung (to A.S.). Back

2 Address correspondence and reprint requests to Dr. Jörg Zwirner, Department of Immunology, Georg August University Gottingen, Kreuzbergring 57, D-37075 Gottingen, Germany. E-mail address: jzwirne{at}gwdg.de Back

3 Abbreviations used in this paper: DC, dendritic cell; BmDC, bone marrow-derived DC; C3aR, C3a receptor; C5aR, C5a receptor; C5aRA, C5a receptor antagonist; CD40L, CD40 ligand; CM, complete medium; MIP, macrophage inflammatory protein; MoDC, monocyte-derived DC. Back

Received for publication September 6, 2002. Accepted for publication January 15, 2003.


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 Introduction
 Materials and Methods
 Results
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