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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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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).
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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).

Mentions: Synthetic MIP-3α was tested at concentrations of 10−6 to 10−10 M in chemotaxis assays to characterize the leukocyte populations that responded to this chemokine (Fig. 3). MIP-3α was a potent chemoattractant for T lymphocytes and CD34+-derived DCs, which migrated with typical bell-shaped dose– response curves. The maximal chemotactic activity on T cells was observed at 10−9 M, whereas on CD34+ DCs the potency was 12.5-fold lower. No chemotactic activity was observed with peripheral blood monocyte–derived DCs, monocytes, or neutrophils. We also tested the chemotactic activity of conditioned medium from HEK 293 cells transfected with wild-type MIP-3α (MIP-3α clone 11) or mutant MIP-3α (MIP-3α clone 16). The level of expression of both proteins (estimated from the HPLC peak height) was similar and ∼0.01–0.1 μg/ml. Whereas the media from both transfectants was chemotactic when compared with conditioned media from mock transfectants, the MIP-3α wild type appeared to be at least 2.5-fold more efficacious than the mutant form.


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)

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).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199050&req=5

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).
Mentions: Synthetic MIP-3α was tested at concentrations of 10−6 to 10−10 M in chemotaxis assays to characterize the leukocyte populations that responded to this chemokine (Fig. 3). MIP-3α was a potent chemoattractant for T lymphocytes and CD34+-derived DCs, which migrated with typical bell-shaped dose– response curves. The maximal chemotactic activity on T cells was observed at 10−9 M, whereas on CD34+ DCs the potency was 12.5-fold lower. No chemotactic activity was observed with peripheral blood monocyte–derived DCs, monocytes, or neutrophils. We also tested the chemotactic activity of conditioned medium from HEK 293 cells transfected with wild-type MIP-3α (MIP-3α clone 11) or mutant MIP-3α (MIP-3α clone 16). The level of expression of both proteins (estimated from the HPLC peak height) was similar and ∼0.01–0.1 μg/ml. Whereas the media from both transfectants was chemotactic when compared with conditioned media from mock transfectants, the MIP-3α wild type appeared to be at least 2.5-fold more efficacious than the mutant form.

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