Specific recruitment of antigen-presenting cells by chemerin, a novel processed ligand from human inflammatory fluids

Abstract

Dendritic cells (DCs) and macrophages are professional antigen-presenting cells (APCs) that play key roles in both innate and adaptive immunity. ChemR23 is an orphan G protein-coupled receptor related to chemokine receptors, which is expressed specifically in these cell types. Here we present the characterization of chemerin, a novel chemoattractant protein, which acts through ChemR23 and is abundant in a diverse set of human inflammatory fluids. Chemerin is secreted as a precursor of low biological activity, which upon proteolytic cleavage of its COOH-terminal domain, is converted into a potent and highly specific agonist of ChemR23, the chemerin receptor. Activation of chemerin receptor results in intracellular calcium release, inhibition of cAMP accumulation, and phosphorylation of p42-p44 MAP kinases, through the Gi class of heterotrimeric G proteins. Chemerin is structurally and evolutionary related to the cathelicidin precursors (antibacterial peptides), cystatins (cysteine protease inhibitors), and kininogens. Chemerin was shown to promote calcium mobilization and chemotaxis of immature DCs and macrophages in a ChemR23-dependent manner. Therefore, chemerin appears as a potent chemoattractant protein of a novel class, which requires proteolytic activation and is specific for APCs.

Figure 1.

Purification of the natural ligand of the ChemR23 receptor from human inflammatory fluid. (A) First step HPLC fractionation (Poros column) of human ascitic fluid. The absorbance (AU) and biological activity on ChemR23 (luminescence in an aequorin-based assay, normalized to the ATP response, solid bars) are shown. (B) Third step (cation-exchange column). (C) Fourth step (C18 column). (D) Last step purification of the active compound (C18 column). The x axis is magnified to focus on the region of interest.

Figure 2.

Identification of chemerin as the natural ligand of ChemR23, the chemerin receptor. (A) Monoisotopic peptide mass fingerprinting of the active fraction on a Maldi Q-TOF mass spectrometer after trypsin digestion. (B) Sequences corresponding to selected major peaks of the Maldi Q-TOF mass spectrometer spectrum after trypsin digestion. Peptides 1–7 correspond to tryptic peptides derived from the Tig-2 gene product (prochemerin), whereas peptide 8 is not tryptic and corresponds to the COOH-terminal end of the purified protein. The position of the peptides within this sequence is given. The sequence of peptides in peaks 3, 5, 7, and 8 was confirmed by microsequencing. (C) Amino acid sequence alignment of human and mouse (Protein Data Bank accession no. AK002298) preprochemerin and human cathelicidin FALL39 precursor. Amino acid identities as compared with human preprochemerin are boxed. The signal peptides (predicted for mouse preprochemerin) are in bold lowercase characters and cysteines are in bold. Cleaved COOH-terminal peptides are in bold italics and underlined (predicted by analogy for mouse prochemerin). The location of introns, which interrupt the gene coding sequences between codons, are indicated by arrowheads.

Figure 3.

Pharmacology of the chemerin receptor. (A) SDS/PAGE of human recombinant chemerin expressed in CHO-K1 cells and purified by HPLC. The gel was silver stained and the major band corresponds to a protein of 18 kD. Mass spectrometry analysis demonstrated the cleavage of the six COOH-terminal amino acids in this biologically active protein. (B) Biological activity on chemerinR of human recombinant chemerin (•) and prochemerin (○), using the aequorin assay. (C) Competition binding assay using as tracer an iodinated peptide derived from the chemerin COOH terminus. Competition was performed with the unlabeled peptide (□) or human recombinant chemerin (•). (D) Concentration-action curve of human chemerin in a GTPγ[³⁵S] binding assay, using membranes of CHO/chemerinR cells. (E) Immunodetection of phosphorylated ERK1/2 in CHO/chemerinR cells after stimulation by human recombinant chemerin for 2 min. (F) Kinetics of ERK1/ ERK2 activation after stimulation by 10 nM human chemerin. Each experiment was repeated at least three times.

Figure 4.

Expression of human chemerin and its receptor. (A) Conversion of 100 nM human recombinant prochemerin in conditioned medium from hamster CHO-K1 cells. Conversion rate was estimated by comparing the biological activity with that of the same molar amount of purified processed chemerin. (B and C) Transcripts encoding human chemerinR (B) and prochemerin (C) were amplified by quantitative RT-PCR in a set of human tissues and cell populations. PBMC, peripheral blood mononuclear cells; iDC, immature DCs. (D and E) The expression of chemerinR was analyzed by FACS^® in immature (solid line) and mature DCs (gray area) after stimulation by LPS (D) or CD40L (E), using the 1H2 monoclonal antibody (IgG2A). Control labeling (dotted line) was made with an antibody of the same isotype. (F) ChemerinR expression on macrophages was monitored using the 1H2 (thick solid line) and 4C7 (thin solid line) monoclonal antibodies. Control labeling (dotted line) was made with an antibody of the same isotype.

Figure 5.

Biological activity of chemerin on primary cells. (A) Inhibition of the functional response of CHO-K1 cells expressing the chemerinR (aequorin assay) by the 4C7 anti-chemerinR monoclonal antibody. The cells were preincubated for 30 min at room temperature with various amounts of the 4C7 antibody before stimulation by 10 nM recombinant chemerin. The data were normalized according to the response in the absence of antibody (100%) and in the absence of agonist (0%). (B) Chemotaxis of human immature DCs by recombinant chemerin. Results are expressed as the mean ± SD (n = 3) and are representative of three donors. (C) 10 pM chemerin-induced DC migration was inhibited by 3 μg/ml pertussis toxin pretreatment of the cells, as well as by preincubation of the cells with 10 μg/ml of the 4C7 monoclonal antibody. Checkerboard analysis investigates chemotactic versus chemokinetic effects of chemerin on DCs. 10 pM human chemerin was added to the lower and/or upper chamber of the chemotaxis device. 10 nM of the chemokine RANTES was used as a positive control in the experiments. (D) Ca²⁺ flux in monocyte-derived DCs in response to 30 nM recombinant chemerin (arrow). (E) The same experiment after 30 min preincubation of the cells with 10 μg/ml of the 4C7 monoclonal antibody. (F) Chemerin-induced macrophage migration (10 and 100 pM) and its inhibition by 3 μg/ml pertussis toxin pretreatment and 10 μg/ml 4C7 monoclonal antibody. Checkerboard analysis investigates chemotactic versus chemokinetic effects of chemerin on macrophages. (G) Ca²⁺ flux in macrophages in response to 30 nM recombinant chemerin (arrow). (H) The same experiment after 30 min preincubation of the cells with 10 μg/ml of the 4C7 monoclonal antibody.