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

Power CA, Church DJ, Meyer A, Alouani S, Proudfoot AE, Clark-Lewis I, Sozzani S, Mantovani A, Wells TN - J. Exp. Med. (1997)

Bottom Line: In the present study, we have shown that the messenger RNA for MIP-3alpha is expressed predominantly in inflamed and mucosal tissues.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.

View Article: PubMed Central - PubMed

Affiliation: Geneva Biomedical Research Institute, GlaxoWellcome Research and Development, Geneva, Switzerland. CAP15123@ggr.co.uk

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.

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(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.
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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.

Mentions: The expression of MIP-3α messenger RNA (mRNA) was analyzed in human multiple tissue Northern blots (Fig. 2 A). MIP-3α mRNA was highly expressed in lung and in inflammed tissues such as tonsil and appendix. Weaker expression was also detected, predominantly at mucosal sites and in lymphatic tissues: placenta, gall bladder, stomach, thymus, prostrate, testis, small intestine, and colon. RT-PCR analysis of distinct leukocyte populations indicated MIP-3α mRNA present in lung macrophages, DCs, and peripheral blood eosinophils (Fig. 2 B).


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

Power CA, Church DJ, Meyer A, Alouani S, Proudfoot AE, Clark-Lewis I, Sozzani S, Mantovani A, Wells TN - J. Exp. Med. (1997)

(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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199050&req=5

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.
Mentions: The expression of MIP-3α messenger RNA (mRNA) was analyzed in human multiple tissue Northern blots (Fig. 2 A). MIP-3α mRNA was highly expressed in lung and in inflammed tissues such as tonsil and appendix. Weaker expression was also detected, predominantly at mucosal sites and in lymphatic tissues: placenta, gall bladder, stomach, thymus, prostrate, testis, small intestine, and colon. RT-PCR analysis of distinct leukocyte populations indicated MIP-3α mRNA present in lung macrophages, DCs, and peripheral blood eosinophils (Fig. 2 B).

Bottom Line: In the present study, we have shown that the messenger RNA for MIP-3alpha is expressed predominantly in inflamed and mucosal tissues.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.

View Article: PubMed Central - PubMed

Affiliation: Geneva Biomedical Research Institute, GlaxoWellcome Research and Development, Geneva, Switzerland. CAP15123@ggr.co.uk

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.

Show MeSH
Related in: MedlinePlus