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Originally published online as doi:10.1189/jlb.1005586 on May 30, 2006

Published online before print May 30, 2006
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(Journal of Leukocyte Biology. 2006;80:342-349.)
© 2006 by Society for Leukocyte Biology

Differential regulation of chemokine production by Fc{gamma} receptor engagement in human monocytes: association of CCL1 with a distinct form of M2 monocyte activation (M2b, Type 2)

Marina Sironi*,1, Fernando O. Martinez{dagger},1, Daniele D’Ambrosio{ddagger}, Marco Gattorno§, Nadia Polentarutti*,1, Massimo Locati{dagger}, Andrea Gregorio§, Andrea Iellem{ddagger}, Marco A. Cassatella, Jo Van Damme||, Silvano Sozzani**, Alberto Martini§, Francesco Sinigaglia{ddagger}, Annunciata Vecchi*,1 and Alberto Mantovani*,{dagger},1,2

* Department of Immunology and Cell Biology, Mario Negri Institute for Pharmacological Research, Milan, Italy;
{dagger} Institute of General Pathology, Medical Faculty, University of Milan, Italy;
{ddagger} BioXell, Milan, Italy;
§ II Division of Pediatrics, University of Genoa, Italy;
Department of Pathology, University of Verona, Italy;
|| DLaboratory of Molecular Immunology, Rega Institute for Medical Research, Leuven, Belgium; and
** Department of Biotechnology, Division of General Pathology and Immunology, University of Brescia, Italy

2Correspondence: Istituto Clinico Humanitas, Via Manzoni, 56, 20089, Rozzano, Italy. E-mail: alberto.mantovani{at}humanitas.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CC chemokine ligand 1 (CCL1; I-309) is a CC chemokine that interacts with CC chemokine receptor 8, which is preferentially expressed in polarized T helper cell type 2 and Tc2 cells, in eosinophils, and in T regulatory cells. The present study, prompted by transcriptional profiling of human monocytes undergoing different forms of activation, was designed to characterize the production of CCL1 in monocytes compared with the production of other chemokines (CCL2, CCL22, and CCL18) differentially regulated by distinct activation signals. Lipopolysaccharide (LPS), interferon-{gamma} (IFN-{gamma}), interleukin (IL)-1ß, tumor necrosis factor {alpha}, IL-4, IL-13, IL-10, IL-6, IL-18, and combinations thereof did not induce CCL1 production in monocytes, and some of these signals stimulated production of reference chemokines. Induction of CCL1 in monocytes required engagement of Fc receptor for immunoglobulin G (Fc{gamma}R)II and exposure to IL-1ß or LPS. This combination of stimuli results in a form of M2 (M2b, Type 2) macrophage activation. Fc{gamma}R engagement also induced CCL22 and amplified its stimulation by IL-4. In contrast, Fc{gamma}R stimulation inhibited the IL-10- and LPS-mediated induction of CCL18. IL-10, IL-4, and IFN-{gamma} inhibited induction of CCL1 by Fc{gamma}R ligation and IL-1ß. CCL1 was present in synovial fluids and macrophages in juvenile idiopathic arthritis. Thus, regulation of CCL1 in human monocytes is unique, with an obligate requirement of Fc{gamma}R engagement and costimulation by signals (IL-1ß and LPS), which use the myeloid differentiation primary-response protein 88 adaptor protein. Thus, CCL1 is a CC chemokine with a unique pattern of regulation associated with a distinct form of M2 (Type 2, M2b) monocyte activation, which participates in macrophage-dependent regulatory circuits of innate and adaptive immunity.

Key Words: IL-1 • MyD88 • CCR8 • Th2


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemokines are small, chemotactic cytokines that play a key role in leukocyte migration under normal and pathological conditions [1 2 3 ]. The spectrum of action of chemokines is generally dictated by differential expression of cognate receptors on distinct leukocyte subsets or stages of differentiation and activation. Polarized CD4+ T helper cell type 1 (Th1) and Th2 cells express distinct sets of chemokine receptors that regulate their trafficking at sites of inflammation. Schematically, fully polarized Th2 cells express preferentially CC chemokine receptor 3 (CCR3), CCR4, and CCR8, whereas Th1 cells express preferentially CCR5 and CXC chemokine receptor 3. T regulatory cells (Treg) have a chemokine receptor repertoire (CCR4 and CCR8) similar to Th2 cells [4 5 6 ]. Regulation of agonist production by interleukin (IL)-4 and interferon-{gamma} (IFN-{gamma}), master cytokines of polarized responses, generally mirrors receptor expression [7 ].

CCR8 is expressed at high levels in polarized Th2 cells and in Treg cells, and these cells migrate in response to the appropriate agonist CC chemokine ligand 1 (CCL1; I-309). Bronchus-infiltrating Th2 cells in patients with asthma express high levels of CCR8, and their numbers correlate with disease severity [8 ]. Accordingly, CCR8 gene-targeted mice show impaired Th2 cytokine production and eosinophil recruitment during Th2-mediated responses [9 ], although this finding has been disputed in a different, deficient strain [10 ]. CCR8 can also be expressed in eosinophils [11 , 12 ], and passive immunization against CCL1 reduces eosinophil recruitments in allergic airway disease. The CCR8/CCL1 axis has also been implicated in cutaneous homing of T cells [13 ] and in the protection against glucocorticoid-induced thymocyte death [14 , 15 ]. Transient expression of CCR8 has been reported recently in monocytes differentiating to dendritic cells (DC) [16 ]. In spite of the strong association of CCR8 with Th2 and Treg circuits, little is known about the regulation of CCL1 production [17 ].

Mononuclear phagocytes are a major source of inflammatory chemokines, including CCL1 [17 ], and they are part of regulatory circuits of polarized responses [18 19 20 21 ]. Based on the expression and function of CCR8, we hypothesized that its cognate ligand CCL1 be induced by cytokines involved in polarized Th2 responses and regulatory circuits (e.g., IL-4, IL-13, IL-10, transforming growth factor-ß). It is unexpected that we observed that regulation of CCL1 in human monocytes is unique, with an obligate requirement of Fc receptor for immunoglobulin G (IgG; Fc{gamma}R) engagement and costimulation by myeloid differentiation primary-response protein 88 (MyD88) using signals [lipopolysaccharide (LPS), IL-1]. Recently, we proposed [19 ] M2 as a generic name for the various forms ("alternatively activated" sensu strictu; Type II; Mø2; M2) of macrophage activation other than the classic M1, and three forms of M2 have been well-defined: M2a (where "a" also stands for alternative), induced by IL-4 or IL-13; M2b, induced by exposure to IC and agonists of Toll-like receptors (TLRs) or IL-1 receptor; and M2c, induced by IL-10 and glucocorticoid hormones. Thus, production of CCL1 is associated with an M2b (Type 2) monocyte activation program [19 , 22 ] and is part of mononuclear phagocyte-mediated regulatory circuits of innate and adaptive immunity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Pyrogen-free saline (SALF, Bergamo, Italy); phosphate-buffered saline (PBS; BioWhittaker, Walkersville, MD); RPMI 1640, L-glutamine, and Ficoll Hypaque (Biochrom, Berlin, Germany); aseptically collected fetal calf serum (FCS; HyClone Laboratories, Logan, UT); sodium butyrate, bovine serum albumin (BSA), and human IgG purified from normal human serum (Sigma Chemical Co., St. Louis, MO); and polyinosinic:polycytidylic acid (poly I:C; Amersham, UK) were all reagents and contained <0.125 endotoxin units/ml, as determined by the Limulus amebocyte lysate assay (BioWhittaker). LPS from Escherichia coli strain 055:B5 was obtained from Difco Laboratories (Detroit, MI), and LPS, from Salmonella abortus equi S-form (ultra pure, TLR grade), was obtained from Alexis (San Diego, CA). IL-1ß was a gift of Dompé Research Centre (L’Aquila, Italy). Schering-Plough Research Institute (Dardilly, France) donated IL-4, IL-6, and IL-10; IL-13 was a gift from Sanofi Elf Bio Recherches (Labège, France); and IL-18 and IFN-{gamma} were from PeproTech (Rocky Hill, NJ). Neutralizing antibodies to CCL1 and IL-10 were from R&D Systems (Minneapolis, MN), and anti-CD16 (Clone LNK16), -CD32 (Clone AT10), and -CD64 (Clone 10.1) were from Serotec (Oxford, UK).

Cell preparation and stimulation
Human peripheral blood monocytes were isolated from buffy coats from healthy donors through the courtesy of Transfusion Center of General Hospital (Desio, Milan, Italy), under protocols approved by the board of the local ethical committee, by Ficoll, and 46% Percoll gradients [23 ]. Monocytes were resuspended at 106/ml in RPMI-1640 medium supplemented with 10% FCS and cultured in 0.2 ml in 96 flat-well plates (Falcon®, Becton Dickinson, Franklin Lakes, NJ) in the presence or absence of the stimuli reported and their combinations. For Fc{gamma}R engagement, wells were coated with human IgG (20 µg/0.2 ml/well in PBS) for 2 h and washed three times with warm PBS before use. Supernatants were collected after 24 h. Macrophages were generated by culturing monocytes (1.5x106/3 ml medium as above) with 100 ng/ml macrophage-colony stimulating factor for 5 days. Cells were recovered with 2 mM EDTA and cultured with the different stimuli as monocytes. CCL1, CCL2, CCL22, and CCL18 were measured by specific enzyme-linked immunosorbent assay (ELISA) purchased by R&D Systems following the supplier’s instructions.

CCR8 transfectants and chemotaxis
L1.2 cells (a mouse pre-B cell line) were cultured in complete medium at 1 x 106/ml. Cells (5x105) were transfected with 3 µg human (h)CCR8 cDNA, a kind gift of Dr. Monica Napolitano (Instituto Dermopatico dell’Immocolata IDI-IRCCS, Rome, Italy) or 3 µg hCCR4 cDNA and 20 µg Lipofectamine reagent (Life Technologies Inc., Grand Island, NY). Forty-eight hours after transfection, cells were cultured in complete medium supplemented with 0.8 mg/ml G418 (Life Technologies Inc.). L1.2 cells, stably expressing hCCR8 and hCCR4, were selected by chemotaxis to CCL1 and CCL17, respectively, and then confirmed by binding with 125I-labeled chemokines. Chemotaxis assay was performed using 5 µm pore polycarbonate filters of a 24-well Transwell chamber (Corning Costar Corp., Cambridge, MA). L1.2-hCCR8 and L1.2-hCCR4 cells were cultured for 16 h with 5 mM sodium butyrate, washed, and resuspended at the concentration of 2 x 106/ml in RPMI 1640 with 0.5% BSA. The supernatants (0.6 ml) from different cell cultures were added to the bottom chamber of the Transwell, 0.1 ml of the cell suspension was added to the top chamber, and plates were incubated for 3 h at 37°C with 5% CO2. After recovery of the cells in the lower compartment of the Transwell, the number of migrated cells was measured with a flow cytometer by 60 s acquisition at a flow rate of 60 µl/min. For the precleaning experiment, 2 ml supernatant was incubated for 2 h at 4°C with 2 µg monoclonal antibody (mAb) anti-CCL1 or isotype-matched control coupled to 25 µl protein G sepharose beads (Pharmacia, Uppsala, Sweden), which were eliminated by centrifugation, and supernatants were tested in chemotaxis.

Real-time polymerase chain reaction (PCR)
Real-time PCR was performed using the SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA) with forward and reverse primers at a final concentration of 300 nM [glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers were used at 200 nM], in a sample volume of 25 µl. Primers were designed using ABI Prism Primer Express 2.0 software (Applied Biosystem) from mRNA sequences submitted to GenBank (accession number M57502) and were: forward TGT TGC TTC TCA TTT GCG G and reverse CTC TTT GCC TCT CTT CAG CTT G. The reaction conditions were as follows: 2 min at 50°C (1 cycle); 10 min at 95°C (1 cycle); and 15 s at 95°C plus 1 min at 60°C (40 cycles). Gene-specific PCR products were measured continuously by means of an ABI PRISM 5700 detection system (Perkin Elmer, Norwalk, CT). Samples were normalized using the housekeeping gene GAPDH. Five replicates for each experimental point were performed, and differences were assessed with the two-tailed Student’s t-test. Results are expressed as relative fold of the stimulated group over the control group, the latter used as a calibrator.

Cell preparation for gene chip analysis
Human monocytes were obtained by two-steps gradient centrifugation as described above. An extra step of purification was included using the Monocyte Isolation Kit II (Miltenyi, Biotec). Cells were stimulated with IL-1 for 18 h, in uncoated or IgG-coated wells.

Chemokine gene transcription analysis using high-density oligonucleotide arrays
After cell stimulation, RNA was purified and labeled as described previously [23 ]. Gene expression analysis corresponding to a single blood donor was performed using the high-density oligonucleotide array Human Genome U133A Arrays (HG-U133A, Affymetrix, Santa Clara, CA) containing 18,400 transcripts and variants, including 14,500 well-characterized human genes and variants. Hybridization and array scanning were carried out as described previously [23 ]. Scanned images and raw data were processed using Robust Multiarray Average [24 ]. Based on metadata included in the Affymetrix website (www.affymetrix.com), a mask was created to exclusively access the entries related to chemokines. Data processing and clustering analyses were performed with Microsoft Excel 2000 SR-1 and TMEV [25 ], respectively. Genes with an absolute fold ≥5 were selected as changed, considering as baseline the untreated cells. Genes and experiments were clustered hierarchically using Pearson correlation as distance and average linkage as main parameters.

Juvenile idiopathic arthritis (JIA) patients
Twenty consecutive JIA patients diagnosed according to the International League of Associations for Rheumatology. Durban’s criteria were included in the study [26 ]. The mean age at the time of the study was 10.1 years (range 3.9–24). The mean disease duration was 6.6 years (range 0.6–18.2). All patients had the onset of their disease before the age of 14 years. Twelve patients had a persistent oligoarticular form, and eight patients had an extended oligoarticular form [26 ]. All patients were positive for antinuclear antibodies and negative for rheumatoid factor. Six patients were on Methotrexate, eight patients were on nonsteroidal anti-inflammatory drugs, and six were off of any treatment. At the moment of the study, all patients had active disease at least at one joint and underwent synovial fluid aspiration for steroids injection, which in the same joint in the previous 6 months was considered as an exclusion criterion. Approval for the study was obtained from the Institutional Medical Ethics Review Board.

Immunohistochemistry
Biopsies were obtained from the inflamed synovium of the knee joint of two JIA patients using direct vision, low-pressure arthroscopy during therapeutic synoviectomy, following informed consent. Tissues were embedded in optical cutting temperature compound (Miles, Elkhart, IN), snap-frozen in liquid nitrogen-cooled isopentane (BDH, Poole, UK), and stored at –80°C. Cryostat sections (6 µ-thick) were mounted on gel-coated slides (Superfrost/Plus, Fisher Scientific, Pittsburgh, PA), wrapped in aluminium foil, and stored at –80°C until use. Before staining, slides were fixed in acetone, air-dried, and treated in 0.3% H2O2 to block endogenous peroxides. Immunohistochemical labeling was performed by a three-step immunoperoxidase technique. Tissue sections were incubated overnight at 4°C with the anti-CCL1 mAb (Clone 35305, R&D Systems) or for 30 min at room temperature with anti-CD68 mAb (Clone KP1, DakoCytomation, Carpinteria, CA) and anti-CD3 polyclonal antibody (DakoCytomation). Sections were subsequently reacted for 30 min at room temperature with anti-mouse Ig antibody conjugated to the peroxidase-labeled dextran polymer (DakoCytomation). Chromogenic diaminobenzidine substrate was applied, and slides were counterstained with Mayer’s hematoxylin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Transcriptional profiling of monocyte M2b activation
In the context of a comprehensive effort aimed at transcriptional profiling human monocytes undergoing different forms of activation and polarization [23 , 27 ], we examined cells exposed to immobilized IgG, to engage Fc{gamma}R, and IL-1. Fc{gamma}R engagement had a profound effect on monocyte gene transcription with 2737-modulated genes. In contrast IL-1 had a limited effect with 1231-regulated genes. The two signals combined resulted in a distinct profile (data online available at www.marionegri.it/profiles). We focused our attention on chemokines, as the two signals elicited a distinct spectrum of these chemoattractants, which included CCL1. As shown in Figure 1 , IL-1 augmented the expression of CXC chemokine ligand 10 (CXCL10; 5.9-fold), whereas Fc{gamma}R engagement stimulated CCL5 (12.4-fold). When monocytes were stimulated with IL-1 and immobilized IgG, dramatic up-regulation of CCL1 (1477-fold) and to a lesser degree, CXCL3 (891-fold) and CCL20 (466-fold) was observed. Given the association of the CCL1 cognate receptor CCR8 with Th2 cells, Treg cells, and eosinophils [4 , 5 , 11 , 12 ] and the limited information on CCL1 regulation in monocytes [17 ], we decided to further characterize CCL1 production in mononuclear phagocytes.


Figure 1
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  		Figure 1. Hierarchical clustering of chemokine genes in monocytes stimulated by Fc{gamma}R engagement and IL-1. Samples were clustered using Pearson correlation; each row corresponds to a gene of interest, and each column represents the relative level of expression of the gene in a given sample. Red indicates a high level of expression of mRNA, and green indicates a low level of expression. Dendrogram links genes according to their expression similarity.

 
Production of CCL1 and reference chemokines
To explore the regulation of CCL1 in a systematic way, in a first series of experiments, we tested the ability of LPS and a wide range of cytokines (IFN-{gamma}, tumor necrosis factor {alpha}, IL-1, IL-4, IL-6, IL-10, IL-13, IL-18) and combinations thereof to induce CCL1 in human monocytes with uniformly negative results (data not shown). Figure 2A only represents a selection of stimuli and combinations tested. Under the same experimental conditions, monocytes responded to stimuli with production of reference chemokines. So, IFN-{gamma}, LPS, and IL-1 induced production of CCL2 (Fig. 2A) [28 ]. IL-4 (Fig. 2B) and IL-13 (not shown) stimulated the release of CCL22, as described previously [7 , 27 ]. It is interesting that production of CCL22 was induced synergistically by IL-4 and IL-1 (Fig. 2B) . As expected, IL-10 as well as IL-4 stimulated the production of CCL18 (Fig. 2C) . Moreover, IL-4 and LPS synergized in terms of CCL18 induction.


Figure 2
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  		Figure 2. Chemokine production by monocytes cultured with inflammatory stimuli or cytokines and their combinations. Monocytes were cultured for 24 h, and chemokines were measured in the supernatants by ELISA. (A) CCL2/monocyte chemoattractant protein-1 (MCP-1). (B) CCL22/macrophage-derived chemokine (MDC). (C) CCL18/pulmonary and activation-regulated chemokine (PARC). Concentrations of the stimuli were: IFN-{gamma} 500 U/ml; IL-1ß, IL-10, and IL-4 20 ng/ml; LPS 100 ng/ml. Results are mean values ± SE from four donors for CCL2 and two donors for CCL22 and CCL18.

 
Figure 3 shows that Fc{gamma}R engagement per se did not induce appreciable levels of CCL1 in monocytes. However, when Fc{gamma}R stimulation was associated with LPS or IL-1, consistent production of CCL1 was observed (Fig. 3A) . CCL1 was first detectable in monocyte supernatants 12 h and 24 h after stimulation with IL-1 and LPS, respectively (Table 1 ). Monocytes exposed to IgG and IL-1 or LPS for 24 h (14 donors) produced 2.94 + 0.81 and 0.44 + 0.11 ng/ml CCL1, respectively. The costimulation of CCL1 in concert with Fc{gamma}R engagement was restricted to IL-1 and LPS, in that no effect was observed when IFN-{gamma}, IL-4, IL-10, and the series of agents mentioned above was used (Fig. 3B) . The restriction to IL-1 and LPS for CCL1 production suggested a role of MyD88 in signaling. To investigate this point further, a LPS ultrapure preparation, and poly I:C, a non-MyD88 TLR stimulus, were tested. As shown in Table 1 , LPS and ultrapure LPS induced the same CCL1 production with the same kinetics, suggesting that the effects seen with LPS were not caused by contaminants activating multiple TLRs. Moreover, no CCL1 production was seen when poly I:C was used in the same experimental conditions (Table 1) . CCL1 protein production was associated with transcript induction as assessed by PCR analysis (Fig. 3C) . Blocking anti-Fc{gamma}R mAb was used to actually assess the relative contribution of different Fc{gamma}Rs. As shown in Figure 4 , anti-CD32 caused a marked inhibition of CCL1 production, whereas anti-CD16 and anti-CD64 had no effect.


Figure 3
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  		Figure 3. Production of CCL1 and reference chemokines by monocytes cultured with immune and inflammatory stimuli. Monocytes were cultured for 24 h in the presence of LPS or IL-1ß (A), IFN-{gamma}, IL-4, or IL-10 (B) in plates untreated (open bars) or pretreated with IgG (shaded bars). Stimuli and numbers of donors are as in Figure 2 . CCL1 results are from 14 donors. Results are mean values ± SE. (C) Quantitative real-time PCR of induction and regulation of CCL1 mRNA expression in the absence (open bars) and in the presence (shaded bars) of IgG and IL-1 (18 h).

 

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  		Table 1. Regulation of CCL1 Production by Fc{gamma}R Engagement

 

Figure 4
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  		Figure 4. Role of Fc{gamma}Rs in CCL1 production. Monocytes were cultured as in Figure 3 with the addition of blocking mAb to CD16, CD32, and CD64. Results are reported as percentage of CCL1 produced in the presence of an isotype (IgG1)-matched, irrelevant mAb. Results reported are mean values ± SE from four donors, stimulated with IL-1 (open bars) or LPS (shaded bars). **, P < 0.01, by Student’s t-test versus monocytes treated with an irrelevant antibody (Ab).

 
In a limited series of experiments (three donors), CCL1 production by macrophages was evaluated. The basal production (0.143±0.101 ng/ml) was increased dramatically by Fc{gamma}R engagement (3.147±1.732 ng/ml) in the absence of IL-1 or LPS, suggesting that the requirement of costimuli for CCL1 production depends on the level of mononuclear phagocyte maturation.

Differential regulation of chemokine production by Fc{gamma}R engagement
The effect of engagement of Fc{gamma}R on chemokine production has not been investigated systematically. As shown in Figure 3 , Fc{gamma}R stimulation per se induced production of CCL2 and CCL22 but not of CCL18. The levels of induction for CCL22 (5.20±0.94 ng/ml) were comparable with those induced by IL-4 (4.00±0.31 ng/ml). When combined, IL-4 and Fc{gamma}R stimulation had an additive effect on CCL22 production (13.4±2.5 ng/ml). Fc{gamma}R engagement also amplified the IFN-{gamma}-induced CCL2 production. In contrast, Fc{gamma}R engagement suppressed the induction of CCL18 by IL-10 and LPS.

We then investigated how selected cytokines influenced CCL1 production induced by costimulation with IgG and IL-1. As shown in Figure 5 , IFN-{gamma}, IL-10, and IL-4 inhibited the production of CCL1 under these conditions. Significant inhibition of CCL1 production was also present at low cytokine concentration, as shown in Table 1 for IL-4. Inhibition of protein production was associated with decreased levels of CCL1 transcripts (Fig. 3C) .


Figure 5
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  		Figure 5. Regulation by cytokines of CCL1 induced in monocytes by costimulation with immune and inflammatory stimuli. Monocytes were cultured for 24 h in the presence of IL-1ß, IFN-{gamma}, IL-4, or IL-10 and their combination in plates pretreated with IgG. CCL1 levels were measured in the supernatants. Results are mean values ± SE from seven donors.

 
LPS, but not IL-1, induced IL-10 under these conditions and costimulated IL-10 production with Fc{gamma}R engagement (Fig. 6A ). LPS-induced IL-10 participates in the regulation of the amount of CCL1 produced, as demonstrated by the doubling of the CCL1 levels measured when monocytes were stimulated in the presence of anti-IL-10 antibodies (Fig. 6B) .


Figure 6
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  		Figure 6. Effect of endogenous IL-10 on CCL1 production. CCL1 and IL-10 production in supernatants of monocytes cultured in untreated or IgG-pretreated wells and stimulated with LPS or IL-1ß. (A). CCL1 production after LPS or IL-1 stimulation of monocytes cultured in the presence of anti-IL-10 antibodies in IgG-pretreated wells (B). Results are from one representative donor of the four tested.

 
It was important to ascertain whether immunoreactive CCL1 was functional. Supernatants from monocytes exposed to IgG and LPS were tested for chemotactic activity on CCR8-transfected L1.2 cells. Costimulation induced high levels of chemotactic activity, which was abolished by an anti-CCL1 antibody. In agreement with immunoreactive protein determinations, addition of IL-4 suppressed the production of chemotactic activity for CCR8 transfectants (Fig. 7 ). Supernatants from monocytes exposed to IL-4 and LPS in the presence of IgG were chemotactic for CCR4 transfectants (not shown), as expected on the basis of CCL22 measurement (Fig. 3) .


Figure 7
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  		Figure 7. Biological activity of monocyte supernatants. Biological activity of monocyte supernatants was evaluated in chemotaxis assay in Transwell using CCR8-transfected L1.2 cells. Specificity was confirmed by the use of specific anti-CCL1 antibodies as detailed in Materials and Methods.

 
CCL1 in rheumatoid arthritis
In an effort to assess whether the regulation of CCL1 in monocytes had any in vivo relevance, patients with juvenile rheumatoid arthritis (RA) were examined, as immune complexes and inflammatory cytokines are present in RA. CCL1 was undetectable in the peripheral blood of normal donors and JIA patients. In contrast, 10/20 JIA patients showed measurable CCL1 with a mean value of 0.074 ± 0.022 ng/ml (Fig. 8A ). At immunohistochemistry, serial sections of synovial tissue showed the expression of CCL1 in correspondence with areas infiltrated with macrophages (CD68-positive cells) in the lining and sublining layer, where CCL1-positive cells were also found in the proximity of T lymphocyte (CD3-positive) aggregates (Fig. 8) . We also examined the synovial fluids of 14 adult patients with RA. In 10 samples, we found CCL1 levels of 0.060 ± 0.007 ng/ml (data not shown).


Figure 8
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  		Figure 8. CCL1 production in JIA patients. Levels of CCL1 were measured in sera from normal donors and JIA patients and in the synovial fluids of JIA patients (A). Expression of CCL1 in inflamed synovial tissue was evaluated by immunohistochemistry; serial sections show the expression of CD68 (B), CCL1 (C), and CD3 (D) in the sublining layer (20x).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results presented here show that CCL1 is produced by human monocytes only when costimulated by Fc{gamma}RII engagement and IL-1 or LPS. A series of eight other cytokines, alone or in combination, was ineffective. Thus, the requirements for CCL1 production in mononuclear phagocytes are clearly different from those of chemokines studied so far and in particular, from CCL2, CCL22, and CCL18 used in the present study as prototype molecules representative of the chemokine repertoire of monocytes undergoing different forms of activation [18 19 20 21 ].

Mononuclear phagocytes are versatile, plastic cells that respond to microenvironmental signals with distinct activation programs [19 , 21 , 29 ]. Classically activated macrophages (M1 cells) following exposure to IFN-{gamma} and LPS acquire potent immunostimulatory and effector function. Following the description of alternative activation by IL-4 (M2a cells) [29 ], various versions of M2 cells have been described [18 19 20 , 22 ]. In general, M2 mononuclear phagocytes tune inflammatory responses and adaptive Th2 immunity, scavenge debris, and promote angiogenesis, tissue repair, and remodeling. In particular, engagement of Fc{gamma}R and LPS results in an M2b or Type 2 [19 , 22 ] IL-12lowIL-10high phenotype. Down-regulation of IL-12 by Fc{gamma}R has been observed in human monocytes, mouse macrophages, and DC [30 31 32 ]. M2b cells promote Th2 responses and protect mice against LPS toxicity [30 , 33 ].

Chemokines are a key component of the different forms of activation of mononuclear phagocytes [19 ]. These molecules are differentially regulated by inducers of various forms of activation, as illustrated by CXCL10, CCL22, and CCL18 (see also Fig. 1 ). The results reported here show that CCL1 has a unique pattern of regulation in human monocytes, with induction restricted to combinations of Fc{gamma}R engagement and LPS or IL-1, and IL-1 and LPS share the MyD88 adaptor protein [34 , 35 ] and components of the signaling cascade. Thus, M2b monocytes have a unique chemokine fingerprint, characterized by CCL1, which when produced by polarized M2b cells, attracts CCR8-bearing Th2 cells, Treg cells, and eosinophils [4 , 5 , 11 , 12 ]. Production of CCL1 by M2b monocytes would therefore represent a circuit of amplification and regulation of polarized inflammation and immunity.


   ACKNOWLEDGEMENTS
 
This work was supported by the European Community (INNOCHEM FP6-518167, DCThera LSHB-CT-2004-512074), by PNR Biotecnologie Avanzate Tema 2, and by the Associazione Italiana per la Ricerca sul Cancro.


   FOOTNOTES
 
Current address: Istituto Clinico Humanitas, via Manzoni, 56, 20089 Rozzano (MI), Italy. Back

Received October 17, 2005; revised March 3, 2006; accepted March 30, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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