Cloning and characterization of a specific receptor for the novel CC chemokine MIP-3alpha from lung dendritic cells

Abstract

Dendritic cells are potent antigen-presenting cells involved in the initiation of immune responses. The trafficking of these cells to tissues and lymph nodes is mediated by members of the chemokine family. Recently, a novel CC chemokine known as MIP-3alpha or liver and activation-regulated chemokine has been identified from the EMBL/GenBank/DDBJ expressed sequence tag database. In the present study, we have shown that the messenger RNA for MIP-3alpha is expressed predominantly in inflamed and mucosal tissues. MIP-3alpha produced either synthetically or by human embryonic kidney 293 cells is chemotactic for CD34(+)-derived dendritic cells and T cells, but is inactive on monocytes and neutrophils. MIP-3alpha was unable to displace the binding of specific CC or CXC chemokines to stable cell lines expressing their respective high affinity receptors, namely CCR1-5 and CXCR1 and CXCR2, suggesting that MIP-3alpha acts through a novel CC chemokine receptor. Therefore, we used degenerate oligonucleotide-based reverse transcriptase PCR to identify candidate MIP-3alpha receptors in lung dendritic cells. Our results show that the orphan receptor known as GCY-4, CKRL-3, or STRL-22 is a specific receptor for MIP-3alpha, and that its activation leads to pertussis toxin-sensitive and phospholipase C-dependent intracellular Ca2+ mobilization when it is expressed in HEK 293 cells.

Figure 1

Amino acid sequence of MIP-3α (clone 11). The signal peptide is underlined. The amino acid deleted in MIP-3α (clone 16) is italicized. Arrows denote the NH₂-terminal amino acid of the mature protein (a), synthetic MIP-3α (b), wild-type MIP-3α (clone 11), and (c) mutant MIP-3α (clone 16).

Figure 2

(A) Northern blot analysis of MIP-3α expression in human tissues. (a) Clontech multiple tissue Northern blots (1 μg poly A+ mRNA/lane); (b) Invitrogen Northern territory blots (20 μg total RNA/ lane). (B) RT-PCR analysis of MIP-3α expression in leukocytes. (Top) MIP-3α; (bottom) glyceraldehyde 3-phosphate dehydrogenase control. Molecular weight markers (1-kb ladder) are shown on the left. Lane 1, lung macrophages; lane 2, T cells; lane 3, monocytes; lane 4, neutrophils; lane 5, eosinophils; lane 6, peripheral blood monocyte–derived DCs; lane 7, NK cells (IL-2–stimulated); lane 8, lung DCs.

Figure 2

(A) Northern blot analysis of MIP-3α expression in human tissues. (a) Clontech multiple tissue Northern blots (1 μg poly A+ mRNA/lane); (b) Invitrogen Northern territory blots (20 μg total RNA/ lane). (B) RT-PCR analysis of MIP-3α expression in leukocytes. (Top) MIP-3α; (bottom) glyceraldehyde 3-phosphate dehydrogenase control. Molecular weight markers (1-kb ladder) are shown on the left. Lane 1, lung macrophages; lane 2, T cells; lane 3, monocytes; lane 4, neutrophils; lane 5, eosinophils; lane 6, peripheral blood monocyte–derived DCs; lane 7, NK cells (IL-2–stimulated); lane 8, lung DCs.

Figure 2

(A) Northern blot analysis of MIP-3α expression in human tissues. (a) Clontech multiple tissue Northern blots (1 μg poly A+ mRNA/lane); (b) Invitrogen Northern territory blots (20 μg total RNA/ lane). (B) RT-PCR analysis of MIP-3α expression in leukocytes. (Top) MIP-3α; (bottom) glyceraldehyde 3-phosphate dehydrogenase control. Molecular weight markers (1-kb ladder) are shown on the left. Lane 1, lung macrophages; lane 2, T cells; lane 3, monocytes; lane 4, neutrophils; lane 5, eosinophils; lane 6, peripheral blood monocyte–derived DCs; lane 7, NK cells (IL-2–stimulated); lane 8, lung DCs.

Figure 3

Chemotaxis of leukocytes in response to synthetic MIP-3α. (a) T cells; (b) monocytes; (c) neutrophils. The response to MIP-3α is indicated by the open circles. As controls for the chemotaxis we used 100 nM each of MCP-1 for T cells and monocytes and IL-8 for neutrophils (closed circles). (d) CD34⁺ DCs (closed circles) and peripheral blood monocyte–derived DCs (open circles). (e) Chemotaxis of T cells in response to conditioned medium from MIP-3α clone 11 transfectants (open circles); MIP-3α clone 16 transfectants (closed circles); and mock transfectants (open squares).

Figure 3

Chemotaxis of leukocytes in response to synthetic MIP-3α. (a) T cells; (b) monocytes; (c) neutrophils. The response to MIP-3α is indicated by the open circles. As controls for the chemotaxis we used 100 nM each of MCP-1 for T cells and monocytes and IL-8 for neutrophils (closed circles). (d) CD34⁺ DCs (closed circles) and peripheral blood monocyte–derived DCs (open circles). (e) Chemotaxis of T cells in response to conditioned medium from MIP-3α clone 11 transfectants (open circles); MIP-3α clone 16 transfectants (closed circles); and mock transfectants (open squares).

Figure 3

Chemotaxis of leukocytes in response to synthetic MIP-3α. (a) T cells; (b) monocytes; (c) neutrophils. The response to MIP-3α is indicated by the open circles. As controls for the chemotaxis we used 100 nM each of MCP-1 for T cells and monocytes and IL-8 for neutrophils (closed circles). (d) CD34⁺ DCs (closed circles) and peripheral blood monocyte–derived DCs (open circles). (e) Chemotaxis of T cells in response to conditioned medium from MIP-3α clone 11 transfectants (open circles); MIP-3α clone 16 transfectants (closed circles); and mock transfectants (open squares).

Figure 4

RT-PCR analysis of DCCR2 expression in leukocytes. Molecular weight markers are shown on the left. Lane 1, lung DCs; lane 2, peripheral blood monocyte–derived DCs; lane 3, CD34⁺ DCs; lane 4, CD4 T cells; lane 5, CD8 T cells.

Figure 5

Dendrogram showing the relationship of DCCR2 with known chemokine receptors and a number of orphan receptors. The EMBL/GenBank/ DDBJ accession numbers for the receptors are CCR1, P32246; CCR2, P41597; CCR3, P51677; CCR4, P51679; CCR5, P51681; V28 (orphan receptor), U28934; U95626 (orphan receptor); TER1 (CCR8), U45983; CXCR1, P25024; CXCR2, P25025; CXCR3, P49682; CXCR4, Q28474; CCR7/EBI1, P32248; DCCR2 is GCY-4, U45984; BLR1 (orphan receptor), X68149; and DARC, Q16570.

Figure 6

Effect of MIP-3α on intracellular free calcium in DCCR2 receptor–expressing HEK 293 cells. HEK 293 cells transiently expressing the DCCR2 receptor were loaded with Fluo-3AM and exposed to either vehicle (buffer) or to 50 nM–1 μM concentrations of MIP-3α. Calcium fluorometry was conducted as described in Materials and Methods. Tracings represent the means of eight experiments conducted in single determinations.

Figure 7

Effect of preincubation with chemokines on MIP-3α–induced calcium responses in DCCR2 receptor–expressing cells. HEK 293 cells transiently expressing DCCR2 were loaded with Fluo-3AM, and exposed for 5 min to either 1 μM IL-8, 1 μM GROα, 1 μM NAP-2, or 500 nM IP-10 (A); 1 μM RANTES, 1 μM MIP-1α, 1 μM MIP-1β, 1 μM MIP-3α, 1 μM MIP-5, 1 μM MCP-1, 1 μM eotaxin, 1 μM fractalkine, 1 μM HCC-1, or 1 μM I-309 (B), or to increasing concentrations of MIP-3α (C), followed by addition of 100 nM MIP-3α. Calcium fluorometry was conducted as described in Materials and Methods. Results represent the mean calcium signal amplitudes of four separate experiments conducted in single determinations. Asterisk, P <0.05 versus effect of 100 nM MIP-3a preceded by vehicle pretreatment, or 1 nM MIP-3α pretreatment for C. The value of 100 = normalized response induced by 100 nM MIP-1α.

Figure 7

Effect of preincubation with chemokines on MIP-3α–induced calcium responses in DCCR2 receptor–expressing cells. HEK 293 cells transiently expressing DCCR2 were loaded with Fluo-3AM, and exposed for 5 min to either 1 μM IL-8, 1 μM GROα, 1 μM NAP-2, or 500 nM IP-10 (A); 1 μM RANTES, 1 μM MIP-1α, 1 μM MIP-1β, 1 μM MIP-3α, 1 μM MIP-5, 1 μM MCP-1, 1 μM eotaxin, 1 μM fractalkine, 1 μM HCC-1, or 1 μM I-309 (B), or to increasing concentrations of MIP-3α (C), followed by addition of 100 nM MIP-3α. Calcium fluorometry was conducted as described in Materials and Methods. Results represent the mean calcium signal amplitudes of four separate experiments conducted in single determinations. Asterisk, P <0.05 versus effect of 100 nM MIP-3a preceded by vehicle pretreatment, or 1 nM MIP-3α pretreatment for C. The value of 100 = normalized response induced by 100 nM MIP-1α.

Figure 7

Effect of preincubation with chemokines on MIP-3α–induced calcium responses in DCCR2 receptor–expressing cells. HEK 293 cells transiently expressing DCCR2 were loaded with Fluo-3AM, and exposed for 5 min to either 1 μM IL-8, 1 μM GROα, 1 μM NAP-2, or 500 nM IP-10 (A); 1 μM RANTES, 1 μM MIP-1α, 1 μM MIP-1β, 1 μM MIP-3α, 1 μM MIP-5, 1 μM MCP-1, 1 μM eotaxin, 1 μM fractalkine, 1 μM HCC-1, or 1 μM I-309 (B), or to increasing concentrations of MIP-3α (C), followed by addition of 100 nM MIP-3α. Calcium fluorometry was conducted as described in Materials and Methods. Results represent the mean calcium signal amplitudes of four separate experiments conducted in single determinations. Asterisk, P <0.05 versus effect of 100 nM MIP-3a preceded by vehicle pretreatment, or 1 nM MIP-3α pretreatment for C. The value of 100 = normalized response induced by 100 nM MIP-1α.

Figure 8

Effect of pertussis toxin treatment, EGTA, PLC inhibition, and Ca²⁺–ATPase inhibition on MIP-3α–induced calcium responses in DCCR2-expressing HEK 293 cells. (A) HEK 293 cells transiently expressing the DCCR2 were treated overnight with vehicle (−Ptx) or 500 nM pertussis toxin (+Ptx), loaded with Fluo-3AM, and incubated with increasing concentrations of MIP-3α. Calcium fluorometry was conducted as described in Materials and Methods. (B) Cells were stimulated with 100 nM MIP-3α in the presence (plus) and absence (minus) of 1.6 mM extracellular EGTA. (C) Cells were exposed for 5 min to 10 μM U-73122 (U73) or 1 μM PMA before addition of 100 nM MIP-3α. (D) Cells were preincubated for 5 min with increasing concentrations of thapsigargin (closed squares) or cyclopiazonate (open circles) before addition of 100 nM MIP-3α. Results in A, C, and D represent the mean maximal calcium signal amplitudes of four separate experiments conducted in single determinations. Asterisk, P <0.05 versus corresponding control. B shows averaged tracings from four experiments. The value of 100 = normalized response induced by 100 nM MIP-1α.

Figure 9

Competition binding of [I¹²⁵] MIP-3α to HEK 293 cells transiently transfected with DCCR2. Binding assays were performed as described in Materials and Methods. Open circles denote binding to pcDNA3.1(+) DCCR2 transfectants; closed circles denote binding to pcDNA3.1(+) CAT transfectants.