CCR6, a CC chemokine receptor that interacts with macrophage inflammatory protein 3alpha and is highly expressed in human dendritic cells.
Little is known about the molecular mechanisms underlying this migratory behavior.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.
Affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.
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.
- Dendritic Cells/immunology*/metabolism
- Macrophage Inflammatory Proteins/metabolism*
- Receptors, Chemokine*
- Receptors, Cytokine/genetics*/metabolism*
- Amino Acid Sequence
- Base Sequence
- CHO Cells
- Chromosome Mapping
- Cloning, Molecular
- DNA, Complementary/genetics
- Gene Expression
- Hybrid Cells
- Molecular Sequence Data
- Polymerase Chain Reaction
- RNA, Messenger/genetics/metabolism
- Receptors, CCR6
- Recombinant Proteins/genetics/metabolism
- Sequence Homology, Amino Acid
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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.
Various direct and indirect approaches were used to assess the distribution of BN-1 mRNA. A Northern blot containing mRNA from a variety of organs and tissues showed an ∼3.5-kb message predominantly in spleen, and to a lesser extent in thymus, testis, small intestine, and peripheral blood (Fig. 3 A, top left). In addition, the spleen showed two smaller transcripts of ∼2.7 and 1.7 kb. mRNAs from various lymphoid and hematopoietic cell lines (TF-1, Jurkat, MRC5, JY, and U937; Fig 3 A, top right) were negative for BN-1 expression. For cell lines and tissues where limited amounts of mRNA precluded direct Northern analysis, expression was determined by the extent of hybridization among the gel-fractionated population of cDNA inserts from libraries made from those cells. BN-1 expression was examined first in 19 cDNA libraries made from various cells of lymphoid lineage (Fig. 3 A, bottom). BN-1 cDNA was present in one library made from resting PBMCs, consistent with the observation of Zaballos et al. for expression of the CKR-L3 orphan cDNA in CD4 and CD8 cells (22), and of the STRL22 orphan in PBLs (23). Interestingly, however, a matched PBMC library made after the cells were activated with anti-CD3 antibody and PMA showed no BN-1 cDNAs, which was further notable by their absence from virtually every other library made from T cell lines and clones (in various states of activation and anergy), pooled B cells, and NK cells (Fig. 3 A, bottom). Resting human splenocytes contain BN-1 cDNA (Fig. 3 A, bottom, Splen), but a matched library made after activation of splenocytes with anti-CD40 and IL-4 (Splen Act) showed diminished levels of BN-1 cDNA. Thus it appears that BN-1 may not be abundantly expressed in the lymphoid lineage, or that its expression is downregulated with cellular activation or growth in lymphocyte cultures.