CCR6, a CC chemokine receptor that interacts with macrophage inflammatory protein 3alpha and is highly expressed in human dendritic cells

Abstract

Dendritic cells initiate immune responses by ferrying antigen from the tissues to the lymphoid organs for presentation to lymphocytes. Little is known about the molecular mechanisms underlying this migratory behavior. We have identified a chemokine receptor which appears to be selectively expressed in human dendritic cells derived from CD34+ cord blood precursors, but not in dendritic cells derived from peripheral blood monocytes. When stably expressed as a recombinant protein in a variety of host cell backgrounds, the receptor shows a strong interaction with only one chemokine among 25 tested: the recently reported CC chemokine macrophage inflammatory protein 3alpha. Thus, we have designated this receptor as the CC chemokine receptor 6. The cloning and characterization of a dendritic cell CC chemokine receptor suggests a role for chemokines in the control of the migration of dendritic cells and the regulation of dendritic cell function in immunity and infection.

Figure 1

Sequence homology of the BN-1–coding region with other chemokine receptors. The deduced 374–amino acid sequence encoded by the BN-1 cDNA was compared to other human and viral chemokine receptors using the multiple sequence alignment program Pileup of the Wisconsin EGCG DNA analysis software package. Shown here is the BN-1 amino acid sequence aligned with the human CXCR2 and CCR4. The positions of the hydrophobic membrane spanning regions TM1–TM7 are indicated by bars above the sequence. Amino acids identical between BN-1 and either CXCR2 or CCR4 are boxed.

Figure 2

The BN-1 gene maps to chromosome 6. (A) PCR analysis of hamster, mouse, human and rodent–human hybrid cell line genomic DNAs (samples 1-20 Biosmap; BIOS Labs., New Haven, CT) using BN-1–specific primers BN1 and BN2. The lane marked Blank is the result of PCR in the absence of template, lanes M1 and M2 contain DNA molecular weight markers. The same genomic DNA samples gave positive PCR signals of the expected size with a second pair of BN-1–specific PCR primers, BN3 and BN4 (see Materials and Methods). (B) The result of PCR analysis of the same genomic DNA samples with human TNF-α promoter primers. The assignment of the BN-1 gene to chromosome 6 was confirmed by PCR analysis of a panel of chromosome 6 deletion and translocation hybrids (data not shown). PCR analysis of the Genebridge 4 radiation hybrid panel with BN1 and BN2 primers gave the following data vector: 12202021002210000101200020000000000000000112010000100 0010200011000020210100001020010100000001. The BN-1 data vector was compared to the WICGR human genome radiation hybrid map using the Whitehead Institute/Massachusetts Institute of Technology Center for Genome Research automapper version 1.0 (http//:www-genome.wi.mit.edu/). The BN-1 STS was placed on chromosome 6, 3.36 centiRay from the framework STS D6S1008 with a lod score >3.0.

Figure 3

Analysis of BN-1 gene expression. (A) Northern blot analysis of BN-1 expression in RNA prepared from the various human tissues and cell lines (top); sizes of RNA markers (in kilobases) are indicated in the left margin. (Bottom) A Southern blot analysis of BN-1 in cDNA libraries prepared from the human PBL and primary cell T, B, and NK cell cultures. PBMC, human peripheral blood mononuclear cells; PBMC Act, the same PBMC stimulated with anti-CD3 and PMA for 2, 6, and 12 h and pooled; Mot 72 and Mot 81, human Th0 T cell clones. Act, Stimulation with anti-CD3 and anti-CD28 for 2, 6, and 12 h and pooled; and Anergic, stimulation with a specific peptide rendering the cells nonresponsive to antigen stimulation. HY06 and HY93 are human Th1 and Th2 T cell clones, respectively, with activation and anergy treatments as above except for the peptide specificity; B cell pool, a collection of EBV cell lines; Splen, total human splenocytes, resting; Splen Act, the same population stimulated with anti-CD40 and IL-4 for 2, 6, and 16 h and pooled; NK pool, a pool of primary NK cell clones; NK Act 6h, the same pool stimulated 6 h with PMA and ionomycin; NK B1, a single primary human NK cell clone; NK Act 6h, that clone activated as above. Probing replicate blots with human β actin cDNA gave readily detectable ∼2.0-kb species in all lanes (data not shown). References upon request. (B) Southern blot of BN-1 distribution (top) in cDNA libraries made from monocytes and DCs. U937, human monocyte cell line. Human elutriated monocytes have been stimulated as follows: LPS/IFNγ, cultured in the presence of these activators and blocking antibodies for IL-10 for 1, 2, 6, 12, and 24 h and pooled; LPS/IFNγ/IL10, the same pool without blocking antibodies to IL-10; LPS 1 h and LPS 6 h, monocytes stimulated for 1 and 6 h with LPS, respectively; CD34-derived DC, 30 and 70% DCs are CD1a⁺ DCs derived from CD34⁺ human cord blood stem cells by growth in GM-CSF and TNF-α, with the percent CD1a⁺ cells determined by FACs^® over time in culture. The cultures were 70% CD1a⁺ after 12 d. 70% CD1a ⁺ act 1 h and act 6 h, the same cultured DC stimulated with PMA and ionomycin for 1 and 6 h, respectively. Mono-derived DC, DCs derived from human elutriated monocytes by growth in GM-CSF and IL-4 for 5 and 10 d as listed; GM/IL4/LPS act, 10-d cultures stimulated for 6 h with LPS; GM/IL4/IL1+TNF, are 10-d monocyte-derived DCs stimulated with IL-1α and TNF-α for 4 and 16 h and pooled. CD34-derived DC, sorted, DCs derived from CD34 cells as described that were then sorted on the basis on the cell surface expression of the markers listed: 95% CD1a⁺, DC derived from CD1a-sorted cells (Langerhans-like); CD1a⁺/CD14⁺, DC derived from CD14-sorted cells (dermal/interstitial). (Bottom) The same blot stripped and reprobed with the human CCF-18/MIP-1γ-chemokine. In all cases, the ladder effect represents different sizes of cDNA inserts in the library ranging from ∼0.6 to 3.5 kbp for BN-1. The two predominant CCF-18/MIP-1γ cDNAS are ∼0.7 and 1.3 kbp. (C) PCR analysis of unamplified mRNA from DC to confirm BN-1 distribution. Lane 1, CD34⁺ cord blood cells cultured 12 d in GM-CSF and TNF-α; lane 2, CD34-derived DCs stimulated with PMA and ionomycin; lane 3, CD34-derived DC purified by FACS^® sorting for 98% CD1a⁺ expression; lanes 4–8, various cultures of monocyte-derived DCs (cultured in GM-CSF + IL-4 for 8 d) under conditions of stimulation as shown; + Ctl, positive control amplification using 1 pg BN-1 plasmid as starting substrate; − Ctl, same reaction with identical reagents in the absence of the plasmid substrate. Elutriated monocytes (not shown) were also negative. Each lane is representative of at least three independent experiments.

Figure 3

Analysis of BN-1 gene expression. (A) Northern blot analysis of BN-1 expression in RNA prepared from the various human tissues and cell lines (top); sizes of RNA markers (in kilobases) are indicated in the left margin. (Bottom) A Southern blot analysis of BN-1 in cDNA libraries prepared from the human PBL and primary cell T, B, and NK cell cultures. PBMC, human peripheral blood mononuclear cells; PBMC Act, the same PBMC stimulated with anti-CD3 and PMA for 2, 6, and 12 h and pooled; Mot 72 and Mot 81, human Th0 T cell clones. Act, Stimulation with anti-CD3 and anti-CD28 for 2, 6, and 12 h and pooled; and Anergic, stimulation with a specific peptide rendering the cells nonresponsive to antigen stimulation. HY06 and HY93 are human Th1 and Th2 T cell clones, respectively, with activation and anergy treatments as above except for the peptide specificity; B cell pool, a collection of EBV cell lines; Splen, total human splenocytes, resting; Splen Act, the same population stimulated with anti-CD40 and IL-4 for 2, 6, and 16 h and pooled; NK pool, a pool of primary NK cell clones; NK Act 6h, the same pool stimulated 6 h with PMA and ionomycin; NK B1, a single primary human NK cell clone; NK Act 6h, that clone activated as above. Probing replicate blots with human β actin cDNA gave readily detectable ∼2.0-kb species in all lanes (data not shown). References upon request. (B) Southern blot of BN-1 distribution (top) in cDNA libraries made from monocytes and DCs. U937, human monocyte cell line. Human elutriated monocytes have been stimulated as follows: LPS/IFNγ, cultured in the presence of these activators and blocking antibodies for IL-10 for 1, 2, 6, 12, and 24 h and pooled; LPS/IFNγ/IL10, the same pool without blocking antibodies to IL-10; LPS 1 h and LPS 6 h, monocytes stimulated for 1 and 6 h with LPS, respectively; CD34-derived DC, 30 and 70% DCs are CD1a⁺ DCs derived from CD34⁺ human cord blood stem cells by growth in GM-CSF and TNF-α, with the percent CD1a⁺ cells determined by FACs^® over time in culture. The cultures were 70% CD1a⁺ after 12 d. 70% CD1a ⁺ act 1 h and act 6 h, the same cultured DC stimulated with PMA and ionomycin for 1 and 6 h, respectively. Mono-derived DC, DCs derived from human elutriated monocytes by growth in GM-CSF and IL-4 for 5 and 10 d as listed; GM/IL4/LPS act, 10-d cultures stimulated for 6 h with LPS; GM/IL4/IL1+TNF, are 10-d monocyte-derived DCs stimulated with IL-1α and TNF-α for 4 and 16 h and pooled. CD34-derived DC, sorted, DCs derived from CD34 cells as described that were then sorted on the basis on the cell surface expression of the markers listed: 95% CD1a⁺, DC derived from CD1a-sorted cells (Langerhans-like); CD1a⁺/CD14⁺, DC derived from CD14-sorted cells (dermal/interstitial). (Bottom) The same blot stripped and reprobed with the human CCF-18/MIP-1γ-chemokine. In all cases, the ladder effect represents different sizes of cDNA inserts in the library ranging from ∼0.6 to 3.5 kbp for BN-1. The two predominant CCF-18/MIP-1γ cDNAS are ∼0.7 and 1.3 kbp. (C) PCR analysis of unamplified mRNA from DC to confirm BN-1 distribution. Lane 1, CD34⁺ cord blood cells cultured 12 d in GM-CSF and TNF-α; lane 2, CD34-derived DCs stimulated with PMA and ionomycin; lane 3, CD34-derived DC purified by FACS^® sorting for 98% CD1a⁺ expression; lanes 4–8, various cultures of monocyte-derived DCs (cultured in GM-CSF + IL-4 for 8 d) under conditions of stimulation as shown; + Ctl, positive control amplification using 1 pg BN-1 plasmid as starting substrate; − Ctl, same reaction with identical reagents in the absence of the plasmid substrate. Elutriated monocytes (not shown) were also negative. Each lane is representative of at least three independent experiments.

Figure 3

Analysis of BN-1 gene expression. (A) Northern blot analysis of BN-1 expression in RNA prepared from the various human tissues and cell lines (top); sizes of RNA markers (in kilobases) are indicated in the left margin. (Bottom) A Southern blot analysis of BN-1 in cDNA libraries prepared from the human PBL and primary cell T, B, and NK cell cultures. PBMC, human peripheral blood mononuclear cells; PBMC Act, the same PBMC stimulated with anti-CD3 and PMA for 2, 6, and 12 h and pooled; Mot 72 and Mot 81, human Th0 T cell clones. Act, Stimulation with anti-CD3 and anti-CD28 for 2, 6, and 12 h and pooled; and Anergic, stimulation with a specific peptide rendering the cells nonresponsive to antigen stimulation. HY06 and HY93 are human Th1 and Th2 T cell clones, respectively, with activation and anergy treatments as above except for the peptide specificity; B cell pool, a collection of EBV cell lines; Splen, total human splenocytes, resting; Splen Act, the same population stimulated with anti-CD40 and IL-4 for 2, 6, and 16 h and pooled; NK pool, a pool of primary NK cell clones; NK Act 6h, the same pool stimulated 6 h with PMA and ionomycin; NK B1, a single primary human NK cell clone; NK Act 6h, that clone activated as above. Probing replicate blots with human β actin cDNA gave readily detectable ∼2.0-kb species in all lanes (data not shown). References upon request. (B) Southern blot of BN-1 distribution (top) in cDNA libraries made from monocytes and DCs. U937, human monocyte cell line. Human elutriated monocytes have been stimulated as follows: LPS/IFNγ, cultured in the presence of these activators and blocking antibodies for IL-10 for 1, 2, 6, 12, and 24 h and pooled; LPS/IFNγ/IL10, the same pool without blocking antibodies to IL-10; LPS 1 h and LPS 6 h, monocytes stimulated for 1 and 6 h with LPS, respectively; CD34-derived DC, 30 and 70% DCs are CD1a⁺ DCs derived from CD34⁺ human cord blood stem cells by growth in GM-CSF and TNF-α, with the percent CD1a⁺ cells determined by FACs^® over time in culture. The cultures were 70% CD1a⁺ after 12 d. 70% CD1a ⁺ act 1 h and act 6 h, the same cultured DC stimulated with PMA and ionomycin for 1 and 6 h, respectively. Mono-derived DC, DCs derived from human elutriated monocytes by growth in GM-CSF and IL-4 for 5 and 10 d as listed; GM/IL4/LPS act, 10-d cultures stimulated for 6 h with LPS; GM/IL4/IL1+TNF, are 10-d monocyte-derived DCs stimulated with IL-1α and TNF-α for 4 and 16 h and pooled. CD34-derived DC, sorted, DCs derived from CD34 cells as described that were then sorted on the basis on the cell surface expression of the markers listed: 95% CD1a⁺, DC derived from CD1a-sorted cells (Langerhans-like); CD1a⁺/CD14⁺, DC derived from CD14-sorted cells (dermal/interstitial). (Bottom) The same blot stripped and reprobed with the human CCF-18/MIP-1γ-chemokine. In all cases, the ladder effect represents different sizes of cDNA inserts in the library ranging from ∼0.6 to 3.5 kbp for BN-1. The two predominant CCF-18/MIP-1γ cDNAS are ∼0.7 and 1.3 kbp. (C) PCR analysis of unamplified mRNA from DC to confirm BN-1 distribution. Lane 1, CD34⁺ cord blood cells cultured 12 d in GM-CSF and TNF-α; lane 2, CD34-derived DCs stimulated with PMA and ionomycin; lane 3, CD34-derived DC purified by FACS^® sorting for 98% CD1a⁺ expression; lanes 4–8, various cultures of monocyte-derived DCs (cultured in GM-CSF + IL-4 for 8 d) under conditions of stimulation as shown; + Ctl, positive control amplification using 1 pg BN-1 plasmid as starting substrate; − Ctl, same reaction with identical reagents in the absence of the plasmid substrate. Elutriated monocytes (not shown) were also negative. Each lane is representative of at least three independent experiments.

Figure 4

Chemokine specificity of the receptor encoded by BN-1. (A) A sorted population of transfected CHO cells stably expressing BN-1 protein containing an NH₂-terminal Flag epitope (CHO-FlagBN-1) showing intensity of anti-Flag mAb staining relative to wild-type CHO cells. (B) Panel of purified recombinant or synthetic chemokines assayed for intracellular calcium mobilizing activity in various BN-1 transfectants. At 100 nM final concentration − indicates no detectable intracellular calcium mobilization in any of the transfectants (n >3), whereas ++ denotes a robust response in all transfectants (CHO, 3T3, HEK293) bearing BN-1. (C) Dose response of MIP-3α in the induction of intracellular calcium in CHO-FlagBN-1 cells. CHO wild-type cells are shown as a control. (D) Binding and homologous competition of radiolabeled MIP-3α to BN-1–bearing cells. (Inset) Scatchard transformation of the binding data to reveal a Kd of ∼0.1 nM.