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, 186 (6), 825-35

Cloning and Characterization of a Specific Receptor for the Novel CC Chemokine MIP-3alpha From Lung Dendritic Cells

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Cloning and Characterization of a Specific Receptor for the Novel CC Chemokine MIP-3alpha From Lung Dendritic Cells

C A Power et al. J Exp Med.

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.

Figures

Figure 1
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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.

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References

    1. Steinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol. 1991;9:271–296. - PubMed
    1. Schall, T.J. 1994. The Chemokines. In The Cytokine Handbook. A. Thompson, editor. Academic Press, New York. 419–460.
    1. Kelner GS, Kennedy J, Bacon KB, Kleyensteuber S, Largaespada DA, Jenkins NA, Copeland NG, Bazan JF, Moore KW, Schall TJ. Lymphotactin: a cytokine that represents a new class of chemokine. Science (Wash DC) 1994;266:1395–1399. - PubMed
    1. Bazan JF, Bacon KB, Hardiman G, Wang W, Soo K, Rossi D, Greaves DR, Zlotnik A, Schall TJ. A new class of membrane-bound chemokine with a CX3C motif. Nature (Lond) 1997;385:640–644. - PubMed
    1. Tashiro K, Tada H, Heilker R, Shirozu M, Nakano T, Honjo T. Signal sequence trap: a cloning strategy for secreted proteins and type I membrane proteins. Science (Wash DC) 1993;261:600–603. - PubMed

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